OMP85 Proteins of Neisseria Gonorrhoeae and Neisseria Meningitidis, Compositions Containing Same and Methods of Use Thereof

ABSTRACT

Nucleic acid and amino acid sequences of the Omp85 proteins of  N. gonorrhoeae  and  N. meningitidis , and fragments thereof are useful in vaccine compositions, therapeutic compositions and diagnostic compositions for use in the prevention, treatment and diagnosis of non-symptomatic gonococcal infection or symptomatic disease and non-symptomatic meningococcal infection and symptomatic disease. Antibodies are developed to these proteins and also useful in the compositions and methods described herein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/606,618, filed Jun. 26, 2003, now pending, which is a continuation ofU.S. patent application Ser. No. 09/994,192, filed Nov. 26, 2001, nowU.S. Pat. No. 6,610,306, issued Aug. 26, 2003, which is a continuationof U.S. patent application Ser. No. 09/177,039, filed Oct. 22, 1998, nowabandoned. All priority applications cited herein are expresslyincorporated by reference.

This invention was made with government support under grant Nos. AI21235and AI37777, awarded by the National Institutes of Health. Thegovernment has certain rights in this invention.

BACKGROUND OF THE INVENTION

This invention relates generally to the cloning and identification ofnovel outer membrane proteins of several strains of Neisseria, and morespecifically to proteins useful in the prevention, therapy and/ordiagnosis of infection and diseases in mammals caused by these strains.

The pathogenic Neisseriae cause several important non-symptomaticinfections and symptomatic diseases in humans. Neisseria gonorrhoeae isthe agent of non-symptomatic gonococcal infection or symptomaticdisease, i.e., gonorrhea. Neisseria meningitidis is the agent of arapidly progressive spinal meningitis, which may also have anon-symptomatic infective stage. The surfaces of such pathogens providecrucial interfaces for interactions between the pathogen and the host.Many bacterial virulence factors are outer membrane proteins, andsurface exposed proteins are the primary targets recognized and attackedby the host's immune system. Thus, the role of outer membrane proteinsis of particular importance in understanding the pathogenesis of theseorganisms. The most abundant and immunodominant outer membrane proteinsof the pathogenic Neisseriae have been studied extensively (Sparling P.F. et al, Clin. Invest., 89: 1699-1705 (1992)). For example, it is knownthat the immunodominant components of the gonococcal surface areantigenically variant, suggesting that this organism is capable ofadapting to varying host environments while avoiding host immuneresponses. Although the major gonococcal surface proteins have beenextensively studied, little is known about less abundant proteins andtheir contributions to pathogenesis.

Two-dimensional electrophoresis (IEF and SDS-PAGE) of labeled, e.g.,radioiodinated or biotinylated, gonococcal surface proteins suggestedthat numerous (>20) of the less abundant gonococcal outer membraneproteins remained uncharacterized (unpublished observations). Amongthese might be proteins which play an important role in infection.

For example, surface-exposed outer membrane proteins of othermicroorganisms, e.g., Haemophilus influenzae D15 surface antigen(D-15-Ag) and the Pasteurella multocida Oma87, have been found to beuseful in eliciting antibodies that were protective against infectiouschallenge in animal models. The Omp85-like D-15-Ag was conserved in bothnon-typeable and typeable strains of H. influenzae and was recognized byconvalescent patient sera; affinity-purified anti-D-15-Ag serum wasprotective in the rat pup model (Thomas, W. R., et al, Infect. Immun.,58:1909-1913 (1990); Flack, F. S. et al, Gene, 156:97-99 (1995)]). H.influenzae serotypes a-f, nontypeable H. influenzae and Haemophilusparainfluenzae all expressed proteins similar to the D-15-Ag, asdemonstrated by immunoblot analysis. Antibodies to recombinant D-15-Agprotected against H. influenzae type b and type a bacteremia in theinfant rat model (Loosmore, S. M. et al, Infect. Immun., 65:3701-3707(1997)).

Like H. influenzae D-15-Ag, the Oma87 of P. multocida was highlyconserved among strains and was recognized by protective antibody; itwas present in all 16 serotypes of P. multocida and was recognized byconvalescent animal sera. Antibodies raised to recombinant Oma87 wereprotective against homologous challenge in the mouse model (Ruffolo, C.G. et al., Infect. Immun., 64:3161-3167 (1996)). Despite the severalpublications describing the immunological properties of D-15-Ag andOma87, the function of these proteins remains unknown.

There remains a need in the art for the development of proteins fromNeisseriae which are useful in research, diagnosis and treatment of theinfections, especially non-symptomatic infections, and the diseasescaused by these pathogens.

SUMMARY OF THE INVENTION

In one aspect, the invention provides an isolated outer membrane proteinof N. gonorrhoeae having an apparent molecular weight of 85 kDa andcharacterized by an amino acid sequence of SEQ ID NO: 2, a fragment, ananalog or a homolog thereof. In another aspect, the invention provides anucleic acid sequence encoding the Omp85 of N. gonorrhoeae or a fragmentthereof.

In still another aspect, the invention provides a nucleic acid moleculecomprising a nucleic acid sequence encoding the Omp85 of N. gonorrhoeaeor a fragment thereof under the control of suitable regulatory sequenceswhich direct expression of said Omp85 protein or fragment in a selectedhost cell.

In yet a further aspect, the invention provides a host cell transformedwith the above described nucleic acid molecule.

In still a further aspect, the invention provides a method ofrecombinantly expressing the Omp85 of N. gonorrhoeae or a fragmentthereof comprising the steps of culturing a recombinant host celltransformed with a nucleic acid sequence encoding said protein orfragment under conditions which permit expression of said protein orpeptide.

In another aspect, the invention provides a method for preparing anOmp85 protein of N. gonorrhoeae or fragment thereof comprisingchemically synthesizing said protein or fragment.

In yet another aspect, the invention provides a diagnostic reagentcomprising a nucleic acid sequence encoding Omp85 of N. gonorrhoeae,isolated from cellular materials with which it is naturally associated,a sequence complementary thereto, a fragment thereof, a sequence whichhybridizes thereto under stringent conditions, an allelic variantthereof, a mutant thereof, or a sequence encoding Omp85 or a fragmentthereof fused to a sequence encoding a second protein, and a detectablelabel which is associated with said sequence.

In still another aspect, the invention provides an isolated antibodywhich is directed against Omp85 of N. gonorrhoeae or a fragment thereof.

In a further aspect, the invention provides an anti-idiotype antibodyspecific for the antibody described above.

In another aspect, the invention provides a diagnostic reagentcomprising the antibody or anti-idiotype antibody described above and adetectable label.

In yet another aspect, the invention provides a vaccine compositioncomprising an effective amount of a Omp85 protein of N. gonorrhoeae, afusion protein or fragment thereof and a pharmaceutically acceptablecarrier. This composition can also include at least one other antigen orfragment thereof.

In another aspect, the invention provides a vaccine compositioncomprising an effective amount of a nucleic acid sequence encoding theOmp85 protein of N. gonorrhoeae, a fusion protein, or a fragment thereofand a suitable nucleic acid delivery vehicle. This vaccine compositionmay also be polyvalent.

In still a further aspect, the invention provides a method ofvaccinating a human or animal against gonococcal infection or diseasecomprising administering to said human or animal a compositioncomprising an effective amount of at least one of the compositionsdescribed above.

Another aspect of the present invention includes a method for diagnosinggonococcal infection or disease in a human or animal comprising thesteps of contacting an Omp85 antigen optionally associated with adetectable label or a homolog thereof with a biological sample from ahuman subject to be diagnosed, wherein the presence of naturallyoccurring antibodies to N. gonorrhoeae in said sample permits theformation of an antigen-antibody complex, and analyzing said sample forthe presence of said complex, which indicates infection with N.gonorrhoeae.

Still another aspect of the invention provides a method for diagnosinggonococcal infection or disease in a human or animal comprising thesteps of: contacting an Omp85 antibody, optionally associated with adetectable label, with a biological sample from a human subject to bediagnosed, wherein the presence of naturally occurring N. gonorrhoeaeOmp85 in said sample permits the formation of an antigen-antibodycomplex, and analyzing said sample for the presence of said complex,which indicates infection with N. gonorrhoeae.

Yet a further aspect of the invention provides a method for diagnosinggonococcal infection or disease in a human or animal comprising thesteps of: employing a nucleic acid sequence encoding all or a portion ofan Omp85 antigen or an Omp85 antibody, optionally associated with adetectable label, as a probe which, when in contact with a biologicalsample from a human subject to be diagnosed, enables detection ofinfection by hybridization or amplification of nucleic acid sequences ofN. gonorrhoeae Omp85 in said sample.

Yet a further aspect of the invention includes a therapeutic compositionuseful in treating humans or animals with non-symptomatic gonococcalinfection or symptomatic disease comprising at least one anti-N.gonorrhoeae Omp85 antibody and a suitable pharmaceutical carrier.

In still another aspect, the invention includes a method for treatingnon-symptomatic gonococcal infection or symptomatic disease in amammalian host comprising administering an effective amount of thetherapeutic composition described above.

In yet another aspect, the invention provides a kit for diagnosinginfection with N. gonorrhoeae in a human or animal comprising an Omp85protein or fragment thereof or an anti-Omp85 antibody or a nucleic acidsequence encoding the protein or antibody as described above.

In another aspect, the invention provides a method of identifyingcompounds which specifically bind to Omp85 of N. gonorrhoeae or afragment thereof, comprising the steps of contacting said Omp85 proteinor fragment with a test compound to permit binding of the test compoundto Omp85; and determining the amount of test compound which is bound toOmp85.

In still another aspect, the invention provides a compound identified bythe above method.

In one aspect, the invention provides an isolated outer membrane proteinof N. meningitidis having an apparent molecular weight of 85 kDa andcharacterized by an amino acid sequence of SEQ ID NO: 4, a fragment, ananalog or a homolog thereof. In another aspect, the invention provides anucleic acid sequence encoding the Omp85 of N. meningitidis or afragment thereof.

In still another aspect, the invention provides a nucleic acid moleculecomprising a nucleic acid sequence encoding the Omp85 of N. meningitidisor a fragment thereof under the control of suitable regulatory sequenceswhich direct expression of said Omp 85 or fragment in a selected hostcell.

In yet a further aspect, the invention provides a host cell transformedwith the above described nucleic acid molecule.

In still a further aspect, the invention provides a method ofrecombinantly expressing the Omp85 of N. meningitidis or a fragmentthereof comprising the steps of culturing a recombinant host celltransformed with a nucleic acid sequence encoding said protein orfragment under conditions which permit expression of said protein orpeptide.

In another aspect, the invention provides a method for preparing anOmp85 protein of N. meningitidis or fragment thereof comprisingchemically synthesizing said protein or fragment.

In yet another aspect, the invention provides a diagnostic reagentcomprising a nucleic acid sequence encoding Omp85 of N. meningitidis,isolated from cellular materials with which it is naturally associated,a sequence complementary thereto, a fragment thereof, a sequence whichhybridizes thereto under stringent conditions, an allelic variantthereof, a mutant thereof, or a sequence encoding Omp85 or a fragmentthereof fused to a sequence encoding a second protein, and a detectablelabel which is associated with said sequence.

In still another aspect, the invention provides an isolated antibodywhich is directed against Omp85 of N. meningitidis or a fragmentthereof.

In a further aspect, the invention provides an anti-idiotype antibodyspecific for the antibody described above.

In another aspect, the invention provides a diagnostic reagentcomprising the antibody or anti-idiotype antibody described above and adetectable label.

In yet another aspect, the invention provides a vaccine compositioncomprising an effective amount of a Omp85 protein of N. meningitidis, afusion protein or fragment thereof and a pharmaceutically acceptablecarrier. This composition can also include at least one other antigen orfragment thereof.

In another aspect, the invention provides a vaccine compositioncomprising an effective amount of a nucleic acid sequence encoding theOmp85 protein of N. meningitidis, a fusion protein, or a fragmentthereof and a suitable nucleic acid delivery vehicle. This vaccinecomposition may also be polyvalent.

In still a further aspect, the invention provides a method ofvaccinating a human or animal against non-symptomatic meningococcalinfection and symptomatic disease comprising administering to said humanor animal a composition comprising an effective amount of at least oneof the compositions described above in either a pharmaceuticallyacceptable carrier or a nucleic acid delivery system.

Another aspect of the present invention includes a method for diagnosingnon-symptomatic gonococcal infection or symptomatic disease in a humanor animal comprising the steps of contacting an Omp85 antigen optionallyassociated with a detectable label or a homolog thereof with abiological sample from a human subject to be diagnosed, wherein thepresence of naturally occurring antibodies to N. meningitidis in saidsample permits the formation of an antigen-antibody complex, andanalyzing said sample for the presence of said complex, which indicatesinfection with N. meningitidis.

Still another aspect of the invention provides a method for diagnosingnon-symptomatic meningococcal infection and symptomatic disease in ahuman or animal comprising the steps of: contacting an Omp85 antibody,optionally associated with a detectable label, with a biological samplefrom a human subject to be diagnosed, wherein the presence of naturallyoccurring N. meningitidis Omp85 in said sample permits the formation ofan antigen-antibody complex, and analyzing said sample for the presenceof said complex, which indicates infection with N. meningitidis.

Yet a further aspect of the invention provides a method for diagnosingnon-symptomatic meningococcal infection and symptomatic disease in ahuman or animal comprising the steps of: employing a nucleic acidsequence encoding all or a portion of an Omp85 antigen or an Omp85antibody, optionally associated with a detectable label, as a probewhich, when in contact with a biological sample from a human subject tobe diagnosed, enables detection of infection by hybridization oramplification of nucleic acid sequences of N. meningitidis Omp85 in saidsample.

Yet a further aspect of the invention includes a therapeutic compositionuseful in treating humans or animals with non-symptomatic meningococcalinfection and symptomatic disease comprising at least one anti-N.meningitidis Omp85 antibody and a suitable pharmaceutical carrier.

In still another aspect, the invention includes a method for treatingnon-symptomatic meningococcal infection and symptomatic disease in amammalian host comprising administering an effective amount of thetherapeutic composition described above.

In yet another aspect, the invention provides a kit for diagnosinginfection with N. meningitidis in a human or animal comprising an Omp85protein or fragment thereof or an anti-Omp85 antibody or a nucleic acidsequence encoding the Omp85 protein or antibody, as described above.

In another aspect, the invention provides a method of identifyingcompounds which specifically bind to Omp85 of N. meningitidis or afragment thereof, comprising the steps of contacting said Omp85 proteinor fragment with a test compound to permit binding of the test compoundto Omp85; and determining the amount of test compound which is bound toOmp85.

In still another aspect, the invention provides a compound identified bythe above method.

Other aspects and advantages of the present invention are describedfurther in the following detailed description of the preferredembodiments thereof, reference being made to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a photograph of a sodium dodecyl sulfate polyacrylamideelectrophoretic gel (SDS-PAGE) illustrating the identification ofrecombinant Omp85 produced by E. coli DH5a/pOmp85, as described inExample 2. Bacterial cell lysates were separated by SDS-PAGE, stainedwith Coomassie Brilliant blue (CBB) or transferred to membranes andprobed with anti-GC-OM serum. From left to right are E. coli DH5a, E.coli DH5a/pOmp85, and N. gonorrhoeae strain FA19. The location of Omp85is indicated. Prestained molecular mass markers (MW), were indicated inkilodaltons (kDa).

FIGS. 2A-2C illustrate the DNA sequence encoding an open reading frameof the N. gonorrhoeae omp85 (SEQ ID NO: 1) and the corresponding deducedamino acid sequence of Omp85 (Genbank accession #U81959) (SEQ ID NO: 2),which is preceded by an untranslated 5′ sequence (SEQ ID NO: 7), andfollowed by an untranslated 3′ sequence (SEQ ID NO: 8). The nucleotidesequence begins with the termination codon of a preceding open readingframe (ORF) that is similar to that of the H. influenzae hypotheticalprotein HI0918 and ends with the initiation codon of a downstream genesimilar to ompH of S. typhimurium (Kosk P. et al, J. Biol. Chem., 264:18973-18980 (1989)). The nucleotides of the omp85 open reading frame arenumbered on the left. A ribosome binding site (underlined) precedes theinitiation codon of the omp85 ORF. The omp85 ORF was preceded andfollowed by rho-independent transcriptional termination sequences(indicated by lines with arrows). The Omp85 precursor polypeptide wascomposed of 792 amino acids. The amino acid sequence is numbered on theright. A putative signal peptide cleavage site was identified (indicatedby arrowhead) (Von Heijne, G., Nucl. Acids Res., 14:4683-4690 (1986)).Cleavage at this site would produce a mature protein having a predictedmolecular weight of 85,842 Da.

FIG. 3 is a photograph of a Western blot that illustrates theidentification of Omp85 in N. gonorrhoeae strains FA19, FA635, FA1090,JS1, F62 and MS11LosA and N. meningitidis strains MP78, MP3, MP81 and HHby Western blot analysis with anti-GC-OM serum. E. coli DH5a and E. coliDH5a/pOmp85 were used as negative and positive controls. Prestainedmolecular mass markers (MW) were indicated in kDa.

FIG. 4 is a photograph of a Southern blot that illustrates theidentification of omp85 in genomic DNA from N. gonorrhoeae FA19 and N.meningitidis strains MP3, MP73, MP81 and HH digested with restrictionendonucleases (HincII, EcoRI, PstI, ClaI) and probed with a 688 byfragment of gonococcal omp85. This fragment was used as a positivecontrol in the first lane. Molecular weight markers are indicated on theleft in kilobase pairs. E. coli DH5a was used as a negative control.

FIG. 5 illustrates the amino acid sequence of N. meningitidis Omp85(Genbank accession #AF021045) (SEQ ID NO: 4) compared to that of N.gonorrhoeae Omp85 (SEQ ID NO: 2). On the top line is the N. meningitidisOmp85 amino acid sequence. Below it are the amino acids that aredifferent in the N. gonorrhoeae Omp85. Amino acids that are absent inthe gonococcal Omp85 are indicated by stars Amino acids that areidentical in the Omp85 homologs of N. meningitidis, N. gonorrhoeae, H.influenzae (D-15-Ag) and P. multocida (Oma87) are underlined. The aminoacids of the meningococcal Omp85 are numbered on the right.

FIG. 6 is a photograph of a Western blot that illustrates theidentification of Omp85 in N. gonorrhoeae strains FA19, FA635, FA1090,JS1, F62 and MS11LosA and N. meningitidis strains MP78, MP3, MP81 and HHby Western blot analysis with anti-Omp85, as described in Example 7. E.coli DH5a, E. coli DH5a/pOmp85 and E. coli DH5a/pMCOmp85 were used asnegative and positive controls. Prestained molecular mass markers (MW)were indicated in kDa.

FIG. 7A is a photograph of a Western blot that illustrates thedistribution of Omp85 in pathogenic and commensal Neisseriae(relationship areas A, B, C, and D) and related Gram negative bacteria:N. gonorrhoeae FA19 (A), Neisseria pharyngis (A), Neisseria cinerea (A),Neisseria lactamica (B), Neisseria mucosae (B), Neisseria flavescens(C), Neisseria animalis (C), Neisseria denitrificans (C), Moraxellacatarrhalis (D), Klebsiella pneumoniae, Pseudomonas aeruginosa, and N.meningitidis HH (A), as described in Example 7. E. coli DH5a and E. coliDH5a/pOmp85, were used as negative and positive controls. Prestainedmolecular mass markers (MW) were indicated in kDa.

FIG. 7B is a photograph of a Western blot that illustrates thedistribution of Omp85 in N. gonorrhoeae FA19, Salmonella typhimurium,Shigella flexneri, E. coli strains 35150 (enterohemorrhagic—EHEC), 35401(enterotoxigenic—ETEC), 43887 (enteropathogenic—EPEC), 43892(enteroinvasive—EIEC) and N. meningitidis, as described in Example 7. E.coli DH5a and E. coli DH5a/pOmp85 were used as negative and positivecontrols. Prestained molecular mass markers (MW) were indicated in kDa.

FIG. 8 is a bar graph showing the results of a gonococcal cell adherenceassay performed with no antibody (black bars), and with Fab fragmentsprepared from antisera to: MS11 Omp 85, MS11 hyperimmune sera to bovineserum albumin (MS11BSA), MS11 hyperimmune serum to normal rabbit serum(MS11NRS), FA19 Omp 85, FA19BSA and FA19NRS at concentrations of 1, 10and 100 μg/ml (see key). The assay was performed as described in Example8.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel, characterized, outer surfacemembrane proteins, referred to as Omp85, from N. gonorrhoeae and N.meningitidis. These novel antigens, fragments thereof, antibodiesdeveloped thereto, the nucleic acid sequences encoding same, and the useof such antigens, antibodies and nucleic acid sequences providediagnostic, therapeutic and prophylactic compositions and methods forthe treatment or prevention of gonococcal and meningococcal infections,particularly non-symptomatic infections, and diseases.

I. The Omp85 Antigens of the Invention

To identify the previously uncharacterized N. gonorrhoeae outer membraneproteins, an N. gonorrhoeae genomic library was screened with anantiserum raised against purified isolated gonococcal outer membranes.The gonococcal gene, omp85, was identified that encodes a 792 amino acidouter membrane protein, Omp85, of N. gonorrhoeae having an apparentmolecular weight of 85 kDa and characterized by the amino acid sequenceof FIGS. 2A-2C and SEQ ID NO: 2. Omp85 has a typical signal peptide anda carboxyl-terminal phenylalanine characteristic of outer membraneproteins. Southern analysis demonstrated that the omp85 gene was presentas a single copy in N. gonorrhoeae and N. meningitidis. PCRamplification was used to obtain a clone of the N. meningitidis omp85homolog. The genes encoding the N. gonorrhoeae and N. meningitidis Omp85proteins have been cloned and sequenced. The omp85 gene and its productin both N. gonorrhoeae and N. meningitidis are characterized in FIGS. 2and 5 below (SEQ ID NOS: 1-4).

The nucleic acid sequences encoding the Omp85 proteins and thestructures of the proteins themselves are described below. Where in thisspecification, protein and/or DNA sequences are defined by their percenthomologies or identities to identified sequences, the algorithms used tocalculate the percent identities or percent similarities include thefollowing: the Smith-Waterman algorithm (J. F. Collins et al, 1988,Comput. Appl. Biosci., 4:67-72; J. F. Collins et al, Molecular SequenceComparison and Alignment, (M. J. Bishop et al, eds.) In PracticalApproach Series: Nucleic Acid and Protein Sequence Analysis XVIII, IRLPress: Oxford, England, UK (1987) pp. 417), and the BLAST and FASTAprograms (E. G. Shpaer et al, Genomics, 38:179-191 (1996)), includingthe BLAST2 program (S. D. Altschul et al, J. Mol. Biol., 215:403-407(1990)). These references are incorporated herein by reference.

A. Nucleic Acid Sequence

The present invention provides bacterial nucleic acid sequences encodingomp85 sequences of N. gonorrhoeae and N. meningitidis. The nucleic acidsequences of this invention are isolated from cellular materials withwhich they are naturally associated. The DNA sequence of the N.gonorrhoeae omp85 (SEQ ID NO: 1) and the corresponding deduced aminoacid sequence of Omp85 (Genbank accession #U81959) (SEQ ID NO: 2) wereobtained as described in Example 2 and in FIGS. 2A-2C. The nucleotidesequence begins with the termination codon of a preceding ORF that issimilar to that of the H. influenzae hypothetical protein HI0918 andends with the initiation codon of a downstream gene similar to ompH ofS. typhimurium [(Kosk P. et al, J. Biol. Chem., 264: 18973-18980(1989)]). A ribosome binding site precedes the initiation codon of theomp85 ORF. The omp85 ORF was preceded and followed by rho-independenttranscriptional termination sequences. The Omp85 precursor polypeptidewas composed of 792 amino acids. A putative signal peptide cleavage sitewas identified (indicated by arrowhead) (Von Heijne, G., Nucl. AcidsRes., 14:4683-4690 (1986)). Cleavage at this site produces a matureprotein having a predicted molecular weight of 85,842 Da.

The DNA sequence of the N. meningitidis omp85 (SEQ ID NO: 3) and thecorresponding deduced amino acid sequence of Omp85 (Genbank accession#AF021045) (SEQ ID NO: 4) were obtained as described in Example 3. FIG.5 shows the comparison between the sequences of the two Omp85 proteins,as well as the similarities between the Omp85 homologs of N.meningitidis, N. gonorrhoeae, H. influenzae (D-15-Ag) and P. multocida(Oma87).

In addition to the full-length nucleic acid sequences encoding the Omp85proteins provided herein, the specification also includes fragments ofthese omp85 genes. Preferably, such fragments are characterized byencoding a biologically active portion of Omp85, e.g., an epitope.Alternatively, other non-epitopic fragments may be useful as probes indiagnostic or research use. Generally, these oligonucleotide fragmentsare at least 10, or at least 15 consecutive nucleotides in length.However, oligonucleotide fragments of varying sizes may be selected asdesired. Such fragments may be used for such purposes as performingpolymerase chain reaction (PCR), e.g., on a biopsied tissue sample. Forexample, useful fragments of omp85 DNA and corresponding sequencescomprise sequences occurring between nucleotides 2161 through 2208 ofSEQ ID NO: 1 and nucleotides 2161 and 2218 of SEQ ID NO: 3. Other usefulfragments may be readily obtained by one of skill in the art by resortto conventional DNA sequencing techniques applied to the sequencesdisclosed herein.

The DNA sequences of SEQ ID NOS: 1 and 3 permit one of skill in the artto readily obtain the corresponding anti-sense strands of these DNAsequences. Further, using known techniques, one of skill in the art canreadily obtain additional genomic and cDNA sequences which flank theillustrated DNA sequences or the corresponding RNA sequences, asdesired. Similarly the availability of SEQ ID NOS: 1 and 3 of thisinvention permits one of skill in the art to obtain other species Omp85analogs, and fragments thereof, by use of the nucleic acid sequences ofthis invention as probes in a conventional technique, e.g., polymerasechain reaction. Allelic variants of these sequences within a species(i.e., sequences containing some individual nucleotide differences froma more commonly occurring sequence within a species, but whichnevertheless encode the same protein or a protein with the samefunction) such as other variants of Omp85 (SEQ ID NOS: 2 and 4) may alsobe readily obtained given the knowledge of these nucleic acid sequencesprovided by this invention.

The present invention further encompasses nucleic acid sequences capableof hybridizing under stringent conditions (see, J. Sambrook et al,Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring HarborLaboratory (1989)) to the sequences of SEQ ID NOS: 1 and 3, theiranti-sense strands, or biologically active fragments thereof. An exampleof a highly stringent hybridization condition is hybridization at 2×SSCat 65° C., followed by a washing in 0.1×SSC at 65° C. for an hour.Alternatively, an exemplary highly stringent hybridization condition isin 50% formamide, 4×SSC at 42° C. Moderately high stringency conditionsmay also prove useful, e.g., hybridization in 4×SSC at 55° C., followedby washing in 0.1×SSC at 37° C. for an hour. An alternative exemplarymoderately high stringency hybridization condition is in 50% formamide,4×SSC at 30° C.

According to the invention, the nucleic acid sequences may be modified.Utilizing the sequence data of SEQ ID NOS: 1 and 3, it is within theskill of the art to obtain other synthetically or recombinantly-preparedpolynucleotide sequences, or modified polynucleotide sequences, encodingthe full-length proteins or useful fragments of the invention. Forexample, one of skill may employ preferred or “preference” codons forexpression of the sequence in selected host cells; thus SEQ ID NOS: 1and 3 may be modified to contain different nucleotide triplets whichencode the same amino acid as encoded by SEQ ID NOS: 1 and 3. Suchmodifications made at the nucleic acid level include, for example,modifications to the nucleotide sequences which are silent or whichchange the amino acids, e.g. to improve expression or secretion of theprotein. Also included are allelic variations, caused by the naturaldegeneracy of the genetic code.

Also encompassed by the present invention are mutants of the omp85sequences, including 5′, 3′ or internal deletions, which encode proteinsthat substantially retain the antigenicity of the full-length Omp85 orother proteins or fragments. Such truncated, or deletion, mutants may beexpressed by modified nucleic acid sequences for the purpose ofaffecting the activity of the full-length or wild-type protein.

As described in more detail below, these nucleic acid sequences areuseful for a variety of diagnostic, prophylactic and therapeutic uses.Advantageously, the nucleic acid sequences are useful in the developmentof diagnostic probes and antisense probes for use in the detection anddiagnosis of infections, particularly non-symptomatic infection, anddiseases caused by these Neisseriae pathogens and by related pathogensdiscussed above by utilizing a variety of known nucleic acid assays,e.g., Northern and Southern blots, polymerase chain reaction (PCR), andother assay techniques known to one of skill in the art. The nucleicacid sequences of this invention are also useful in the production ofOmp 85 proteins and homologs as well as a variety of fusion or othersynthetic proteins.

The nucleotide sequences of the invention may be readily synthesized ormay be isolated by conventional uses of polymerase chain reaction orcloning techniques such as those described herein and in conventionaltexts such as Sambrook et al, cited above. For example, the nucleic acidsequences of the antigen of this invention may be prepared or isolatedfrom genomic libraries using DNA primers and probes and PCR techniques.These sequences, fragments thereof, modifications thereto and thefull-length sequences may be constructed recombinantly usingconventional genetic engineering or chemical synthesis techniques orPCR, and the like by utilizing the information provided herein. Further,such nucleic acid sequences may be conventionally labeled for diagnosticuse. Alternatively for use as therapeutic or vaccine components, thenucleic acid sequences of this invention may be admixed with a varietyof pharmaceutically acceptable carriers, e.g., saline, liposomes, etc.or incorporated into nucleic acid molecules, e.g., plasmids under theregulatory control of sequences which direct expression of the encodedprotein in a selected host cell. The nucleic acid sequences may also bedelivered to a host as “naked” DNA or in a gene delivery vehicle, suchas a recombinant virus, all as described in detail below.

B. Protein Sequences

The present invention also provides Omp85 proteins and peptides of thisinvention. These proteins are free from association with othercontaminating proteins or materials with which they are found in nature.The Neisseriae Omp85 antigen has a relative molecular mass of 85 kDa asmeasured by Western immunoblot (See Example 2 and FIGS. 2A-2C). In oneembodiment, the invention provides a gonococcal Omp85 antigen (SEQ IDNO:2) polypeptide of about 792 amino acids, with a signal peptide,having a predicted molecular weight of 85,842 daltons.

The meningococcal omp85 was found to encode a 797 amino acid polypeptidewith a predicted molecular weight of 88.5 kDa (FIG. 5). Between aminoacid residues 720 and 745, the menigococcal Omp85 varied substantiallyfrom gonococcal Omp85, including the insertion of five additional aminoacids. The deduced amino acid sequence (SEQ ID NO: 4) of N. meningitidisOmp85 was revealed by sequence analysis to be 95% identical to N.gonorrhoeae Omp85 and 98% similar to gonococcal Omp85 using the BLAST2algorithm.

The similarities of these two Omp85 proteins to proteins of othermicroorganisms provide evidence of an immunological role played by theseproteins, as well as other potential roles. The D-15 protective surfaceantigen (D-15-Ag) of Haemophilus influenzae and the Oma87 of Pasteurellamultocida are the only bacterial proteins, which have been previouslydescribed, that are similar to the Omp85 amino acid sequence (SEQ ID NO:2). This similarity suggested that these proteins played an importantrole in host-pathogen interactions and have an important function inpathogenesis. The importance of these Omp85 proteins in pathogenesis andimmunobiology was demonstrated by the fact that antibody to similarproteins in H. influenzae and P. multocida were protective. Thesimilarities suggest that the Neisseriae Omp85 proteins are likelyimportant immunological targets of the host immune response.

Western blot analysis demonstrated proteins similar to Omp85 in all ofthe Neisseriae tested with anti-Omp85 in three Neisseriae relationshipareas. Area A contains the frank pathogens N. gonorrhoeae and N.meningitidis and opportunistic organisms known to cause severe humandiseases such as N. pharyngis and N. cinerea. Area B contains species,such as N. mucosae and N. lactamica, typically found in the humannasopharynx which are able to cause opportunistic infections indebilitated hosts. Area C consists of commensal/saprophytic organisms,such as N. flavescens, N. animalis and N. denitricans, which generallydo not cause human infections.

All Neisseriae species colonize mucosal surfaces. The presence ofOmp85-like proteins in numerous pathogenic and commensal organismssuggests it may be involved in establishing or maintaining colonization.The identification of Omp85 proteins in a number of pathogenic andcommensal organisms provides evidence that the Omp85 proteins providefunctions involved in establishing or maintaining colonization.

Database searches identified genes in a number of pathogens which encodehypothetical proteins similar to Omp85, including genes in B. abortus,H. pylori, and B. burgdorferi. The proteins encoded by these genes havenot yet been characterized. A search of the Omp85 amino acid sequenceagainst the GenBank data base resulted in the identification of acyanobacterium protein (Kaneto T. et al, DNA _(—) Res., 3: 109-136(1996)) with 35% similarity to the gonococcal Omp85. The cyanobacteriumprotein was named IAP75 because of its similarity to the 75 kDachloroplast import associated protein, IAP75 (Schnell D J et al,Science, 266: 1007-1011 (1994)). The chloroplast IAP75 was located inthe outer membrane of chloroplasts and was one of four outer membranecomponents of a complex that transports polypeptides. This suggestedthat Omp85 might be part of a similar transport complex.

The sequences of other proteins from the chloroplast import associatedcomplex were searched against the Gonococcal Genome Sequencing ProjectData Base (Dyer and Rowe, 1997). Sequences similar to the chloroplastIAP34 protein (Kessler F. et al, Science, 266: 1035-1039 (1994)) wereidentified in the gonococcal genome. IAP34 is believed to be aGTP-binding protein and the sequences of highest similarity to thegonococcal homolog were in regions identified as GTP-binding proteinmotifs. Surface-crosslinkage studies were performed to determine ifOmp85 might participate in a system analogous to the IAP complex (datanot shown). Studies using the reversible crosslinker DTBP, whichcrosslinks proteins that are within 11.9A of each other (Newhall W J. etal, Infect. Immun., 28: 785-791 (1980)), showed that Omp85 crosslinkedwith up to five other outer membrane proteins, one of ˜34 kDa (IAP34homolog) (data not shown). These data, which confirmed Omp85 was exposedon the bacterial surface, support the role for Omp85 of participating ina complex analogous to that of the chloroplast IAP complex. Furthercharacterization of proteins associated with Omp85 in the outer membranemay provide evidence of additional biological functions of these Omp85proteins.

One of skill in the art using conventional techniques, such as PCR, mayreadily use the Omp85 sequences provided herein to identify and isolateother similar proteins. Such methods are routine and not considered torequire undue experimentation, given the information provided herein.

Antigens of this invention may be characterized by immunologicalmeasurements including, without limitation, Western blot, macromolecularmass determinations by biophysical determinations, such asSDS-PAGE/staining, high pressure liquid chromatography (HPLC) and thelike, antibody recognition assays, T-cell recognition assays, majorhistocompatibility complex (MHC) binding assays, and assays to inferimmune protection or immune pathology by adoptive transfer of cells,proteins or antibodies.

The Omp85 outer membrane antigens of this invention (as well as itsnaturally occurring variants or analogs in other species) may beisolated in the form of a complete intact protein, or a polypeptide orfragment thereof. In one embodiment, Omp85 is isolated by immunoblotprocedures according to its respective molecular mass, as describedbelow in the examples. Such isolation provides the antigen in a formsubstantially free from other proteinaceous and non-proteinaceousmaterials of the microorganism. The molecules comprising thepolypeptides and antigens of this invention may be isolated and furtherpurified using any of a variety of conventional methods including, butnot limited to: liquid chromatography such as normal or reverse phase,using HPLC, FPLC and the like; affinity chromatography (such as withinorganic ligands or monoclonal antibodies); size exclusionchromatography; immobilized metal chelate chromatography; gelelectrophoresis; and the like. One of skill in the art may select themost appropriate isolation and purification techniques without departingfrom the scope of this invention.

Alternatively, the amino acid sequences of the proteins of thisinvention may be produced recombinantly following conventional geneticengineering techniques (see e.g., Sambrook et al, cited above and thedetailed description of making the proteins below).

i. Analogs/Modified Antigen

Also included in the invention are analogs, or modified versions, of theOmp85 protein or fragments provided herein. Typically, such analogsdiffer from the specifically identified proteins by only one to fourcodon changes. Examples include polypeptides with minor amino acidvariations from the illustrated partial amino acid sequence of, forexample, the gonococcal Omp85 (SEQ ID NO: 2), in particular,conservative amino acid replacements. Conservative replacements arethose that take place within a family of amino acids that are related intheir side chains and chemical properties. Also provided are homologs ofthe proteins of the invention which are characterized by having at least80% identity with SEQ ID NO:2 or SEQ ID NO: 4. Also included in thisinvention are homologs having at least 85% identity with SEQ ID NO: 2 orSEQ ID NO: 4. Homologs having at least 90% identity with either SEQ IDNO: 2 or SEQ ID NO: 4 are also encompassed by this invention. Homologshaving at least 95% identity with either SEQ ID NO: 2 or SEQ ID NO: 4are also encompassed by this invention. Also provided are homologs ofthe proteins of the invention which are characterized by having at least85% homology with SEQ ID NO:2 or SEQ ID NO: 4. Also included in thisinvention are homologs having at least 90% homology with SEQ ID NO: 2 orSEQ ID NO: 4. Homologs having at least 95% homology with either SEQ IDNO: 2 or SEQ ID NO: 4 are also encompassed by this invention. Homologshaving at least 99% homology with either SEQ ID NO: 2 or SEQ ID NO: 4are also encompassed by this invention. The algorithms used for thesecalculations are identified above. Based on the sequence informationprovided herein, one of skill in the art can readily obtain full-lengthhomologs and analogs from other bacterial species.

An antigen of the present invention may also be modified to increase itsimmunogenicity. For example, the antigen may be coupled to chemicalcompounds or immunogenic carriers, provided that the coupling does notinterfere with the desired biological activity of either the antigen orthe carrier. For a review of some general considerations in couplingstrategies, see Antibodies, A Laboratory Manual, Cold Spring HarborLaboratory, ed. E. Harlow and D. Lane (1988). Useful immunogeniccarriers known in the art, include, without limitation, keyhole limpethemocyanin (KLH); bovine serum albumin (BSA), ovalbumin, purifiedprotein derivative of tuberculin (PPD); red blood cells; tetanus toxoid;cholera toxoid; agarose beads; activated carbon; or bentonite. Usefulchemical compounds for coupling include, without limitation,dinitrophenol groups and arsonilic acid. One of skill in the art mayreadily select other appropriate immunogenic carriers or couplingagents. The antigen may also be modified by other techniques, such asdenaturation with heat and/or SDS.

ii. Fragments/Deletion Mutants

Further encompassed by this invention are additional fragments of theOmp85 polypeptides and peptides identified herein. Such fragments aredesirably characterized by having a biological activity similar to thatdisplayed by the complete protein, including, e.g., the ability toinduce antibodies which can interfere with the binding of the pathogento its cellular targets (see Example 8). These fragments may be designedor obtained in any desired length, including as small as about 5-8 aminoacids in length up to fragments encompassing just short of the entireprotein. Such fragments may represent consecutive amino acids in theprotein sequence or they may represent conformational sites of theprotein. Such a fragment may represent an epitope or conformationalepitope of the protein.

The Omp85 proteins (SEQ ID NOS:2 and 4) of the invention may be modifiedto create deletion mutants, for example, by truncation at the amino orcarboxy termini, or by elimination of one or more amino acids. Deletionmutants are also encompassed by this invention, as are the DNA sequencesencoding them.

In yet another embodiment, the Omp85 peptides or polypeptides of thisinvention may be in the form of a multiple antigenic peptide (“MAP”,also referred to as an octameric lysine core peptide) construct. Such aconstruct may be designed employing the MAP system described by Tam,Proc. Natl. Acad. Sci. USA, 85:5409-5413 (1988). This system makes useof a core matrix of lysine residues onto which multiple copies of thesame protein or peptide of the invention are synthesized as described(D. Posnett et al., J. Biol. Chem., 263(4):1719-1725 (1988); J. Tarn,“Chemically Defined Synthetic Immunogens and Vaccines by the MultipleAntigen Peptide Approach”, Vaccine Research and Developments, Vol. 1,ed. W. Koff and H. Six, pp. 51-87 (Marcel Deblau, Inc., New York 1992)).Each MAP contains multiple copies of only one peptide.

Still other modified fragments of Omp85 may be prepared by any number ofnow conventional techniques to improve production thereof, to enhanceprotein stability or other characteristics, e.g. binding activity orbioavailability, or to confer some other desired property upon theprotein. Other useful fragments of these polypeptides may be readilyprepared by one of skill in the art using known techniques, such asdeletion mutagenesis and expression.

iii. Fusion or Multimeric Proteins and Compositions

The Omp85 protein of the present invention, or fragments of it, may alsobe constructed, using conventional genetic engineering techniques aspart of a larger and/or multimeric protein or protein compositions.Antigens of this invention may be in combination with outer surfaceproteins or other proteins or antigens of other pathogens, such as thoseidentified above, or various fragments of the antigens described hereinmay be in combination with each other. In such combination, the antigenmay be in the form of a fusion protein. The antigen of the invention maybe optionally fused to a selected polypeptide or protein derived fromother microorganisms. For example, an antigen or polypeptide of thisinvention may be fused at its N-terminus or C-terminus to a polypeptidefrom another pathogen or to more than one polypeptide in sequence.Polypeptides which may be useful for this purpose include polypeptidesidentified by the prior art.

Still another fusion protein of this invention is provided by expressingthe DNA molecule formed by the omp85 DNA sequence or a fragment thereoffused to DNA fragments that are homologous (between about 25-95%identity) to Omp85. One example of such a protein comprises the aminoacid sequence of SEQ ID NO: 2 to which is fused amino acid fragmentsthat are up to 95% identical to that sequence, e.g., from SEQ ID NO: 4,or from any of the above-described homologous proteins. These fragmentsmay be inserted in any order and may contain repeated sequences. Thefused fragments may produce a large DNA molecule which expresses aprotein which may stimulate a variety of antibody specificities.

These fusion proteins comprising multiple polypeptides of this inventionare constructed for use in the methods and compositions of thisinvention. These fusion proteins or multimeric proteins may be producedrecombinantly, or may be synthesized chemically. They also may includethe polypeptides of this invention fused or coupled to moieties otherthan amino acids, including lipids and carbohydrates. Further, antigensof this invention may be employed in combination with other vaccinalagents described by the prior art, as well as with other species ofvaccinal agents derived from other microorganisms. Such proteins areuseful in the prevention, treatment and diagnosis of diseases caused bya wide spectrum of Neisseriae isolates.

A protein composition which may be a preferred alternative to the fusionproteins described above is a cocktail (i.e., a simple mixture)containing different Omp85 proteins or fragments, optionally mixed withdifferent antigenic proteins or peptides of other pathogens. Suchmixtures of these proteins or antigenic fragments thereof are likely tobe useful in the generation of desired antibodies to a wide spectrum ofNeisseriae isolates.

iv. Salts

An antigen of the present invention may also be used in the form of apharmaceutically acceptable salt. Suitable acids and bases which arecapable of forming salts with the polypeptides of the present inventionare well known to those of skill in the art, and include inorganic andorganic acids and bases.

II. Methods of Making Antigens and Nucleic Acid Sequences of theInvention

A. Expression In Vitro

To produce recombinant Omp85 or peptide fragments of this invention, theDNA sequences of the invention are inserted into a suitable expressionsystem. Desirably, a recombinant molecule or vector is constructed inwhich the polynucleotide sequence encoding the selected protein, e.g.,Omp85, is operably linked to a heterologous expression control sequencepermitting expression of the protein. Numerous types of appropriateexpression vectors are known in the art for protein expression, bystandard molecular biology techniques. Such vectors are selected fromamong conventional vector types including insects, e.g., baculovirusexpression, or yeast, fungal, bacterial or viral expression systems.Other appropriate expression vectors, of which numerous types are knownin the art, can also be used for this purpose. Methods for obtainingsuch expression vectors are well-known. See, Sambrook et al, MolecularCloning. A Laboratory Manual, 2d edition, Cold Spring Harbor Laboratory,New York (1989); Miller et al, Genetic Engineering, 8:277-298 (PlenumPress 1986) and references cited therein.

Suitable host cells or cell lines for transfection by this methodinclude bacterial cells. For example, the various strains of E. coli,e.g., HB101, MC1061, and strains used in the following examples, arewell-known as host cells in the field of biotechnology. Various strainsof B. subtilis, Pseudomonas, Streptomyces, and other bacilli and thelike are also employed in this method.

Mammalian cells, such as human 293 cells, Chinese hamster ovary cells(CHO), the monkey COS-1 cell line or murine 3T3 cells derived fromSwiss, Balb-c or NIH mice are used. Another suitable mammalian cell lineis the CV-1 cell line. Still other suitable mammalian host cells, aswell as methods for transfection, culture, amplification, screening,production, and purification are known in the art. (See, e.g., Gethingand Sambrook, Nature, 293:620-625 (1981), or alternatively, Kaufman etal, Mol. Cell. Biol., 5(7):1750-1759 (1985) or Howley et al, U.S. Pat.No. 4,419,446). Many strains of yeast cells known to those skilled inthe art are also available as host cells for expression of thepolypeptides of the present invention. Other fungal cells may also beemployed as expression systems. Alternatively, insect cells such asSpodoptera frugipedera (Sf9) cells may be used.

Thus, the present invention provides a method for producing recombinantOmp85 proteins, which involves transfecting, e.g., by conventional meanssuch as electroporation, a host cell with at least one expression vectorcontaining a polynucleotide of the invention under the control of atranscriptional regulatory sequence. The transfected or transformed hostcell is then cultured under conditions that allow expression of theprotein. The expressed protein is recovered, isolated, and optionallypurified from the cell (or from the culture medium, if expressedextracellularly) by appropriate means known to one of skill in the art.

For example, the proteins are isolated in soluble form following celllysis, or extracted using known techniques, e.g., in guanidine chloride.If desired, the proteins or fragments of the invention are produced as afusion protein. Such fusion proteins are those described above.Alternatively, for example, it may be desirable to produce fusionproteins to enhance expression of the protein in a selected host cell,to improve purification, or for use in monitoring the presence of thedesired protein, e.g., Omp85, in tissues, cells or cell extracts.Suitable fusion partners for the proteins of the invention are wellknown to those of skill in the art and include, among others,β-galactosidase, glutathione-S-transferase, poly-histidine and maltosebinding protein.

B. Expression In Vivo

Alternatively, where it is desired that the Omp85 (whether full-lengthor a fragment) be expressed in vivo, e.g., to induce antibodies, oralternatively where the omp85 is to be employed as a DNA vaccine, anappropriate vector for delivery is readily selected by one of skill inthe art. Exemplary vectors for in vivo gene delivery are readilyavailable from a variety of academic and commercial sources, andinclude, e.g., adeno-associated virus (International patent applicationNo. PCT/US91/03440), adenovirus vectors (M. Kay et al, Proc. Natl. Acad.Sci. USA, 91:2353 (1994); S. Ishibashi et al, J. Clin. Invest., 92:883(1993)), or other viral vectors, e.g., various poxviruses, vaccinia,etc. Methods for insertion of a desired gene, e.g., Omp85, and obtainingin vivo expression of the encoded protein, are well known to those ofskill in the art.

III. Antibodies of the Invention

The present invention also provides antibodies capable of recognizingand binding the isolated, or modified, or multimeric antigens of thisinvention, including antibodies derived from mixtures of such antigensor fragments thereof. Certain of the antibodies of this invention may bespecific to the N. gonorrhoeae or N. meningitidis Omp85 proteins, bybinding to epitopes on the proteins which differ from the former speciesto the latter species. For example, an antibody specific for N.gonorrhoeae may bind an epitope on SEQ ID NO: 2 which is not present inSEQ ID NO: 4, or vice versa. Thus, an N. gonorrhoeae Omp85-specificantibody is defined herein as an antibody that binds an Omp85 antigen ofN. gonorrhoeae only. An N. meningitidis Omp85-specific antibody isdefined herein as an antibody that binds an Omp85 antigen of N.meningitidis only. Alternatively, certain antibodies to these proteinsmay bind an epitope present on both the N. gonorrhoeae and N.meningitidis Omp85 proteins. Still other antibodies of this inventionmay bind an epitope on N. gonorrhoeae and N. meningitidis Omp85, and thesame epitope on the other homologous proteins in homologous orheterologous species of bacteria having homologous proteins (describedin Part I, B above). All of these antibodies are encompassed by thisinvention.

These antibodies are useful in diagnosis of gonococcal and meningococcalinfection (non-symptomatic) as well as symptomatic diseases, caused byN. gonorrhoeae, N. meningitidis or other Neisseriae species, and intherapeutic compositions for treating humans and/or animals that testpositive for infection, or, prior to testing, exhibit symptoms of suchdiseases. The antibodies are useful in diagnosis alone or in combinationwith antibodies to other antigens of this invention, as well asantibodies to other known antigens from homologous or completelyheterologous species of microorganism. These antibodies are also usefulin passive vaccine compositions, which vaccines may also be polyvalent,by containing antibodies to antigens of other microorganisms as well asantibodies to the Omp85 proteins of this invention.

The antibodies of this invention are generated by conventional meansutilizing the isolated, recombinant or modified antigens of thisinvention, or mixtures of such antigens or antigenic fragments. Forexample, polyclonal antibodies are generated by conventionallystimulating the immune system of a selected animal or human with theisolated antigen or mixture of antigenic proteins or peptides of thisinvention, allowing the immune system to produce natural antibodiesthereto, and collecting these antibodies from the animal or human'sblood or other biological fluid.

For example, an antibody according to the invention is produced byadministering to a vertebrate host the antigen or antigenic compositionof this invention, e.g., Omp85. Preferably a recombinant version ofOmp85 (rOmp85) or an Omp85 MAP is used as an immunogen. A suitablepolyclonal antibody against the Omp85 antigen may be generated asantisera, such as the Omp85 antisera employed in the examples herein.

Thus, an antibody of the invention is isolated by affinity purifyingantiserum generated during an infection of a mammal, e.g., a mouse, withN. gonorrhoeae or N. meningitidis, using as immunoabsorbant the Omp85antigen identified herein. Similarly, an antibody of the invention isisolated by immunizing mice with a purified, recombinant antigen of thisinvention, or a purified, isolated Omp85 protein of native origin.

Monoclonal antibodies (MAbs) directed against Omp85 are also generated.Hybridoma cell lines expressing desirable MAbs are generated bywell-known conventional techniques, e.g. Kohler and Milstein and themany known modifications thereof. Similarly desirable high titerantibodies are generated by applying known recombinant techniques to themonoclonal or polyclonal antibodies developed to these antigens (see,e.g., PCT Patent Application No. PCT/GB85/00392; British PatentApplication Publication No. GB2188638A; Amit et al., Science,233:747-753 (1986); Queen et al., Proc. Nat'l. Acad. Sci. USA,86:10029-10033 (1989); PCT Patent Application No. WO90/07861; andRiechmann et al., Nature, 332:323-327 (1988); Huse et al, Science,246:1275-1281 (1988)a).

Given the disclosure contained herein, one of skill in the art maygenerate chimeric, humanized or fully human antibodies directed againstOmp85 or antigenic fragments thereof by resort to known techniques bymanipulating the complementarity determining regions of animal or humanantibodies to the antigen of this invention. See, e.g., E. Mark andPadlin, “Humanization of Monoclonal Antibodies”, Chapter 4, The Handbookof Experimental Pharmacology, Vol. 113, The Pharmacology of MonoclonalAntibodies, Springer-Verlag (June, 1994).

Alternatively, the antigens are assembled as multi-antigenic complexes(see, e.g., European Patent Application 0339695, published Nov. 2, 1989)or as simple mixtures of antigenic proteins/peptides and employed toelicit high titer antibodies capable of binding the selected antigen(s)as it appears in the biological fluids of an infected animal or human.

Further provided by the present invention are anti-idiotype antibodies(Ab2) and anti-anti-idiotype antibodies (Ab3). Ab2 are specific for thetarget to which anti-Omp85 antibodies of the invention bind and Ab3 aresimilar to Omp85 antibodies (Ab1) in their binding specificities andbiological activities (see, e.g., M. Wettendorff et al., “Modulation ofanti-tumor immunity by anti-idiotypic antibodies.” in Idiotypic Networkand Diseases, ed. by J. Cerny and J. Hiernaux J, Am. Soc. Microbiol.,Washington D.C.: pp. 203-229, (1990)). These anti-idiotype andanti-anti-idiotype antibodies are produced using techniques well knownto those of skill in the art. Such anti-idiotype antibodies (Ab2) canbear the internal image of Omp85 or fragments thereof and are thususeful for the same purposes as Omp85 or the fragments.

In general, polyclonal antisera, monoclonal antibodies and otherantibodies which bind to the selected antigen (Ab1) are useful toidentify epitopes of Omp85 to separate Omp85 and analogs thereof fromcontaminants in living tissue (e.g., in chromatographic columns and thelike), and in general as research tools and as starting materialessential for the development of other types of antibodies describedabove. Anti-idiotype antibodies (Ab2) are useful for binding the sametarget and thus may be used in place of the original antigen, e.g.,Omp85, to induce an immune response. The Ab3 antibodies are useful forthe same reason the Ab1 are useful. Other uses as research tools and ascomponents for separation of Omp85 from other contaminants, for example,are also contemplated for the above-described antibodies.

For use in diagnostic assays, the antibodies are associated withconventional labels which are capable, alone or in concert with othercompositions or compounds, of providing a detectable signal. Where morethan one antibody is employed in a diagnostic method, the labels aredesirably interactive to produce a detectable signal. Most desirably,the label is detectable visually, e.g. colorimetrically. A variety ofenzyme systems have been described in the art which will operate toreveal a colorimetric signal in an assay. As one example, glucoseoxidase (which uses glucose as a substrate) releases peroxide as aproduct. Peroxidase, which reacts with peroxide and a hydrogen donorsuch as tetramethyl benzidine (TMB) produces an oxidized TMB that isseen as a blue color. Other examples include horseradish peroxidase(HRP) or alkaline phosphatase (AP), and hexokinase in conjunction withglucose-6-phosphate dehydrogenase which reacts with ATP, glucose, andNAD+ to yield, among other products, NADH that is detected as increasedabsorbance at 340 nm wavelength. Other label systems that may beutilized in the methods of this invention are detectable by other means,e.g., colored latex microparticles (Bangs Laboratories, Ind.) in which adye is embedded may be used in place of enzymes to form conjugates withthe antibodies and provide a visual signal indicative of the presence ofthe resulting complex in applicable assays. Still other labels includefluorescent compounds, radioactive compounds or elements. Detectablelabels for attachment to antibodies useful in diagnostic assays of thisinvention may be easily selected from among numerous compositions knownand readily available to one skilled in the art of diagnostic assays.The methods and antibodies of this invention are not limited by theparticular detectable label or label system employed.

IV. Diagnostic Methods and Assays

The present invention also provides methods of diagnosing infections anddiseases caused by infection with N. gonorrhoeae, N. meningitidis orpossibly other species of pathogen which have homologous proteins to theOmp85 proteins of this invention. These diagnostic methods are usefulfor diagnosing humans having non-symptomatic infection or exhibiting theclinical symptoms of gonococcal or meningococcal disease, or possiblyany of the other diseases caused by homologous bacterial species.

In one embodiment, this diagnostic method involves detecting thepresence of naturally occurring anti-Omp85 antibodies which are producedby the infected human or animal patient's immune system in itsbiological fluids, and which are capable of binding to the antigens ofthis invention or combinations thereof. This method comprises the stepsof incubating a Omp85 antigen or antigenic fragment of this inventionwith a sample of biological fluid or tissue from the patient. Antibodiespresent in the fluids as a result of bacterial infection will form anantibody-antigen complex with the antigen. Subsequently the reactionmixture is analyzed to determine the presence or absence of theseantigen-antibody complexes. The step of analyzing the reaction mixturecan comprise detecting a label associated with the Omp85 antigen, orcontacting the reaction mixture with a labeled specific binding partnerfor the antibody or antibody.

In one embodiment of the method, purified antigen, fragment or mixtureof antigens is electro- or dot-blotted onto nitrocellulose paper.Subsequently, the biological fluid (e.g. serum or plasma) is incubatedwith the blotted antigen, and antibody in the biological fluid isallowed to bind to the antigen(s). The bound antibody is then detectedby standard immunoenzymatic methods.

In another embodiment of the method, latex beads are conjugated to theantigen(s) of this invention. Subsequently, the biological fluid isincubated with the bead/protein conjugate, thereby forming a reactionmixture. The reaction mixture is then analyzed to determine the presenceof the antibodies.

In another embodiment, the diagnostic method of the invention involvesdetecting the presence of the naturally occurring Omp85 itself in itsassociation with the Neisseriae pathogen in the biological fluids ortissue of an animal or human infected by the pathogen. This methodincludes the steps of incubating an antibody of this invention (e.g.produced by administering to a suitable human and/or animal an antigenof this invention preferably conventionally labeled for detection) witha biological sample from a human or an animal to be diagnosed. In thepresence of infection of the human or animal patient, anantigen-antibody complex is formed (specific binding occurs).Subsequently, excess labeled antibody is optionally removed, and thereaction mixture is analyzed to determine the presence or absence of theantigen-antibody complex and the amount of label associated therewith.

Assays employing a protein antigen of the invention can be heterogenous(i.e., requiring a separation step) or homogenous. If the assay isheterogenous, a variety of separation means can be employed, includingcentrifugation, filtration, chromatography, or magnetism.

One preferred assay for the screening of blood products or otherphysiological or biological fluids is an enzyme linked immunosorbantassay, i.e., an ELISA. Typically in an ELISA, the isolated antigen(s) ofthe invention is adsorbed to the surface of a microtiter well directlyor through a capture matrix (i.e., antibody). Residual protein-bindingsites on the surface are then blocked with an appropriate agent, such asbovine serum albumin (BSA), heat-inactivated normal goat serum (NGS), orBLOTTO (a buffered solution of nonfat dry milk which also contains apreservative, salts, and an antifoaming agent). The well is thenincubated with a biological sample suspected of containing specificanti-N. gonorrhoeae or N. meningitidis antibody. The sample can beapplied neat, or more often, it can be diluted, usually in a bufferedsolution which contains a small amount (0.1-5.0% by weight) of protein,such as BSA, NGS, or BLOTTO. After incubating for a sufficient length oftime to allow specific binding to occur, the well is washed to removeunbound protein and then incubated with labeled anti-humanimmunoglobulin (a HuIg) or labeled antibodies to other species, e.g.,dogs. The label can be chosen from a variety of enzymes, includinghorseradish peroxidase (HRP), β-galactosidase, alkaline phosphatase, andglucose oxidase, as described above. Sufficient time is allowed forspecific binding to occur again, then the well is washed again to removeunbound conjugate, and the substrate for the enzyme is added. Color isallowed to develop and the optical density of the contents of the wellis determined visually or instrumentally.

Further, MAbs or other antibodies of this invention which are capable ofbinding to the antigen(s) can be bound to ELISA plates. In anotherdiagnostic method, the biological fluid is incubated on theantibody-bound plate and washed. Detection of any antigen-antibodycomplex and qualitative measurement of the labeled MAb are performedconventionally, as described above.

Other useful assay formats include the filter cup and dipstick. In theformer assay, an antibody of this invention is fixed to a sintered glassfilter to the opening of a small cap. The biological fluid or sample (5ml) is worked through the filter. If the antigen is present (i.e., N.gonorrhoeae infection), it will bind to the filter which is thenvisualized through a second antibody/detector. The dipstick assayinvolves fixing an antigen or antibody to a filter, which is then dippedin the biological fluid, dried and screened with a detector molecule.

Other diagnostic assays can employ the omp85 gene sequences or fragmentsof this invention as nucleic acid probes or an anti-sense sequences,which can identify the presence of infection in the biological fluid byhybridizing to complementary sequences produced by the pathogen in thebiological fluids. Such techniques, such as PCR, Northern or Southernhybridizations etc. are well known in the art. For this purpose, thenucleic acid sequences or fragments of this invention may beconventionally labelled by well known techniques, possibly employing oneor more of the labels described above with reference to use of theantibodies, or with labels more suited for attachment to nucleic acids.Selection of such labels is a routine matter and does not limit thisinvention.

It should be understood by one of skill in the art that any number ofconventional protein assay formats, particularly immunoassay formats, ornucleic acid assay formats, may be designed to utilize the isolatedantigens and antibodies or their nucleic acid sequences or anti-sensesequences of this invention for the detection of N. gonorrhoeae or N.meningitidis infection (as well as infection with other bacteriacharacterized by antigens homologous to the Omp85 antigens of thisinvention) in animals and humans. This invention is thus not limited bythe selection of the particular assay format, and is believed toencompass assay formats which are known to those of skill in the art.

V. Diagnostic Kits

For convenience, reagents for ELISA or other assays according to thisinvention may be provided in the form of kits. Such kits are useful fordiagnosing bacterial infection in a human or an animal sample. Such adiagnostic kit contains an antigen of this invention and/or at least oneantibody capable of binding an antigen of this invention, or the nucleicacid sequences encoding such Omp85 antigens or antibodies or theiranti-sense sequences. Alternatively, such kits may contain a simplemixture of such antigens or nucleic acid sequences, or means forpreparing a simple mixture.

These kits can include microtiter plates to which the antigenic proteinsor antibodies or nucleic acid sequences of the invention have beenpre-adsorbed, various diluents and buffers, labeled conjugates for thedetection of specifically bound antigens or antibodies, or nucleic acidsand other signal-generating reagents, such as enzyme substrates,cofactors and chromogens. Other components of these kits can easily bedetermined by one of skill in the art. Such components may includepolyclonal or monoclonal capture antibodies, antigen of this invention,or a cocktail of two or more of the antibodies, purified orsemi-purified extracts of these antigens as standards, MAb detectorantibodies, an anti-mouse or anti-human antibody with indicator moleculeconjugated thereto, an ELISA plate prepared for absorption, indicatorcharts for colorimetric comparisons, disposable gloves, decontaminationinstructions, applicator sticks or containers, and a sample preparatorcup. Such kits provide a convenient, efficient way for a clinicallaboratory to diagnose N. gonorrhoeae or N. meningiditus infection.

VI. Therapeutic and Vaccine Compositions

A. Protein-Containing Therapeutic and Vaccine Compositions

The antigens and antibodies of the invention, alone or in combinationwith other antigens and antibodies of, or directed to, other pathogenicmicroorganisms may further be used in therapeutic compositions and inmethods for treating humans and/or animals with non-symptomaticinfection or symptomatic disease caused by N. gonorrhoeae, N.meningitidis or the other pathogens identified above.

For example, one such therapeutic composition may be formulated tocontain a carrier or diluent and one or more of the anti-Omp 85antibodies of the invention. In compositions containing the Omp85antigen or antibodies thereto, i.e., protein components, suitablepharmaceutically acceptable carriers may be employed that facilitateadministration of the proteins but are physiologically inert and/ornonharmful.

A variety of such pharmaceutically acceptable protein carriers, and/orcomponents suitable for administration therewith may be selected by oneof skill in the art. For example, pharmaceutical carriers include,without limitation, sterile saline, lactose, sucrose, calcium phosphate,gelatin, dextran, agar, pectin, peanut oil, olive oil, sesame oil, andwater. Additionally, the carrier or diluent may include a time delaymaterial, such as glycerol monostearate or glycerol distearate alone orwith a wax. In addition, slow release polymer formulations can be used.Liposomes or liposomal-like vehicles may also be employed.

Optionally, these compositions may also contain conventionalpharmaceutical ingredients, such as preservatives, or chemicalstabilizers. Suitable ingredients which may be used in a therapeuticcomposition in conjunction with the antibodies include, for example,casamino acids, sucrose, gelatin, phenol red, N-Z amine, monopotassiumdiphosphate, lactose, lactalbumin hydrolysate, and dried milk.

Alternatively, or in addition to the antigens or antibodies of theinvention, other agents useful in treating the disease in question,e.g., antibiotics or immunostimulatory agents and cytokine regulationelements, are expected to be useful in reducing or eliminating diseasesymptoms. Such agents may operate in concert with the therapeuticcompositions of this invention. The development of therapeuticcompositions containing these agents is within the skill of one in theart in view of the teachings of this invention.

Additionally, the therapeutic compositions may be polyvalent. Suchcompositions may contain therapeutic components from other bacterial orviral pathogens, e.g., components of bacterial species homologous toNeiserriae or heterologous thereto and/or antigens from adisease-causing virus. Depending upon the compatibility of thecomponents, the Omp 85 antibodies or antigens of this invention may thusbe part of a multi-component therapeutic composition directed at morethan a single disease.

As a further embodiment of this invention, a therapeutic method involvestreating a human or an animal for infection with N. gonorrhoeae or N.meningitidis by administering an effective amount of such a therapeuticcomposition. An “effective amount” of a proteinaceous composition may bebetween about 0.05 to about 1000 μg/ml of an antibody or antigen of theinvention. A suitable dosage may be about 1.0 ml of such an effectiveamount. Such a composition may be administered 1-3 times per day over a1 day to 12 week period.

However, suitable dosage adjustments for protein or nucleic acidcontaining compositions may be made by the attending physician orveterinarian depending upon the age, sex, weight and general health ofthe human or animal patient. Preferably, such a composition isadministered parenterally, preferably intramuscularly or subcutaneously.However, it may also be formulated to be administered by any othersuitable route, including orally or topically. The selection of theroute of delivery and dosage of such therapeutic compositions is withinthe skill of the art.

In another embodiment, the Omp85 antigens, antibodies, and fragments ofthe invention, alone or in combination with other antigens, antibodies,and fragments from other microorganisms, may further be used incompositions directed to induce a protective immune response in asubject to the pathogen. These components of the present invention arealso useful in methods for inducing a protective immune response inhumans and/or animals against infection with N. gonorrhoeae, N.meningitidis or the other pathogens identified above.

In one embodiment, an outer membrane protein antigen-based vaccine forthe prevention of non-symptomatic gonococcal infection or symptomaticdisease, non-symptomatic meningococcal infection and symptomaticdisease, and other diseases in humans and other animals is providedwhich contains an effective amount of the Omp85 antigen of thisinvention, and a pharmaceutically acceptable carrier or diluent. Thisvaccine composition may contain one or more of the isolated,recombinant, modified or multimeric forms of the Omp85 antigen of theinvention, or mixtures thereof. Similarly, salts of the antigenicproteins may be employed in such compositions.

In another embodiment of this invention, a polyvalent vaccinecomposition may include not only the Omp85 antigen or immunogenicfragment thereof, but may also include antigens from otherdisease-causing agents. Such other agents may be antigens from otherNeisseriae strains. Such other agents may be antigens from completelydistinct bacterial pathogens or from viral pathogens. Combinations ofthe antigen(s) of this invention with other antigens or fragmentsthereof are also encompassed by this invention for the purpose ofinducing a protective immune response in the vaccinated subject to morethan a single pathogen. The selection of these other vaccine componentsis not a limitation of the present invention, and may be left to one ofskill in the art.

Such proteinaceous vaccines may include exemplary carriers as describedabove for therapeutic compositions. Optionally, the vaccine compositionsmay optionally contain adjuvants, preservatives, chemical stabilizers,as well as other conventionally employed vaccine additives. Typically,stabilizers, adjuvants, and preservatives are optimized to determine thebest formulation for efficacy in the target human or animal. Suitableexemplary preservatives include chlorobutanol, potassium sorbate, sorbicacid, sulfur dioxide, propyl gallate, the parabens, ethyl vanillin,glycerin, phenol, and parachlorophenol.

With regard to the adjuvant, one or more of the above described vaccinecomponents may be admixed or adsorbed with a conventional adjuvant. Theadjuvant is used to attract leukocytes or enhance an immune response.Such adjuvants include, among others, RIBI adjuvant, mineral oil andwater, aluminum hydroxide, AMPHIGEN adjuvant, ADJUVAX adjuvant, AVRIDINEadjuvant, L121/squalene, D-lactide-polylactide/glycoside, pluronicplyois, muramyl dipeptide, killed Bordetella, and saponins, such as QuilA.

The invention thus also encompasses a prophylactic method entailingadministering to an animal or human an effective amount of such acomposition. The protein antigenic composition is administered in an“effective amount”, that is, an amount of antigen that is effective in aroute of administration to provide a vaccinal benefit, i.e., protectiveimmunity. Suitable amounts of the antigen can be determined by one ofskill in the art based upon the level of immune response desired. Ingeneral, however, the vaccine composition contains between 1 ng to 1000mg antigen, and more preferably, 0.05 μg to 1 mg per ml of antigen.Suitable doses of the vaccine composition of the invention can bereadily determined by one of skill in the art. Generally, a suitabledose is between 0.1 to 5 ml of the vaccine composition. Further,depending upon the human patient or the animal species being treated,i.e. its weight, age, and general health, the dosage can also bedetermined readily by one of skill in the art.

In general, the vaccine can be administered once; and optionallyboosters can be administered periodically thereafter. The vaccine may beadministered by any suitable route. However, parenteral administration,particularly intramuscular, and subcutaneous, is the preferred route.Also preferred is the oral route of administration. Routes ofadministration may be combined, if desired, or adjusted.

In still another vaccine embodiment, the invention includes acomposition which delivers passive protection against infection by thepathogen. For this composition, the antibodies against the Omp85proteins disclosed herein are useful to provide to the subject ashort-term, passive immune protection against infection. These passiveimmunity vaccine compositions may contain antibodies to other pathogensand suitable vaccine additives as described above, e.g., adjuvants, etc.These compositions may be administered in dosages similar to thosedescribed above for the compositions which actively induce immuneprotection in the vaccinated subject.

B. Nucleic Acid Containing Compositions

The nucleic acid sequences or anti-sense sequences of the invention,alone or in combination with other nucleic acid sequences encodingantigens or antibodies of, or directed to other pathogenicmicroorganisms may further be used in therapeutic compositions and inmethods for treating humans and/or animals with the disease caused byinfection with N. gonorrhoeae, N. meningitidis or the other pathogensidentified above. In another embodiment, the nucleic acid sequences ofthis invention, alone or in combination with nucleic acid sequencesencoding other antigens or antibodies from other pathogenicmicroorganisms, may further be used in compositions directed to activelyinduce a protective immune response in a subject to the pathogen. Thesecomponents of the present invention are useful in methods for inducing aprotective immune response in humans and/or animals against infectionwith N. gonorrhoeae, N. meningitidis or the other pathogens identifiedabove.

For use in the preparation of the therapeutic or vaccine compositions,nucleic acid delivery compositions and methods are useful, which areknown to those of skill in the art. The omp85 sequences or fragmentsthereof (or anti-sense sequences as desired) may be employed in themethods of this invention or in the compositions described herein as DNAsequences, either administered as naked DNA, or associated with apharmaceutically acceptable carrier. These sequences provide for in vivoexpression of the Omp85 protein or peptide or provide for production ofanti-sense sequences which can bind to the Omp85 protein in the subjectdue to infection. So-called ‘naked DNA’ may be used to express the Omp85protein or peptide fragment (or anti-sense sequences) in vivo in apatient. See, e.g., J. Cohen, Science, 259:1691-1692 (Mar. 19, 1993); E.Fynan et al., Proc. Natl. Acad. Sci., USA, 90: 11478-11482 (December1993); J. A. Wolff et al., Biotechniques, 11:474-485 (1991) whichdescribe similar uses of ‘naked DNA’, all incorporated by referenceherein. For example, “naked” omp85 DNA (or anti-sense sequences)associated with regulatory sequences may be administered therapeuticallyor as part of the vaccine composition e.g., by injection.

Alternatively, omp85 DNA or anti-sense DNA may be administered as partof a vector or as a cassette containing the Omp85-encoding DNA sequencesor fragments or anti-sense sequences thereof operatively linked to apromoter sequence and other plasmid sequences. Briefly, the DNA encodingthe Omp85 protein (or anti-sense sequence) or desired fragment thereofmay be inserted into a nucleic acid cassette. This cassette may beengineered to contain, in addition to the omp85 sequence to be expressed(or anti-sense sequence), other optional flanking sequences which enableits insertion into a vector. This cassette may then be inserted into anappropriate DNA vector downstream of a promoter, an mRNA leadersequence, an initiation site and other regulatory sequences capable ofdirecting the replication and expression of that sequence in vivo. Thisvector permits infection of vaccinate's cells and expression of theomp85 (or anti-sense sequence) in vivo.

Numerous types of appropriate vectors are known in the art for proteinexpression and may be designed by standard molecular biology techniques.Such vectors are selected from among conventional vector types includinginsects, e.g., baculovirus expression, or yeast, fungal, bacterial orviral expression systems. Methods for obtaining such vectors arewell-known. See, Sambrook et al., Molecular Cloning. A LaboratoryManual, 2d edition, Cold Spring Harbor Laboratory, New York (1989);Miller et al., Genetic Engineering, 8:277-298 (Plenum Press 1986) andreferences cited therein. Recombinant viral vectors, such asretroviruses or adenoviruses, are preferred for integrating theexogenous DNA into the chromosome of the cell.

Also where desired, the regulatory sequences in such a vector whichcontrol and direct expression of the omp85 gene product or anti-sensesequence in the transfected cell include an inducible promoter.Inducible promoters are those which “turn on” expression of the genewhen in the presence of an inducing agent. Examples of suitableinducible promoters include, without limitation, the sheepmetallothionine (MT) promoter, the mouse mammary tumor virus (MMTV), thetet promoter, etc. The inducing agents may be a glucocorticoid such asdexamethasone, for, e.g., the MMTV promoter, or a metal, e.g., zinc, forthe MT promoter; or an antibiotic, such as tetracycline for tetpromoter. Still other inducible promoters may be selected by one ofskill in the art, such as those identified in International patentapplication WO95/13392, published May 18, 1995, and incorporated byreference herein. The identity of the inducible promoter is not alimitation of this invention.

When omp85 nucleic acid sequences or anti-sense sequences are employedas the therapeutic agent or vaccine agent as ‘naked DNA’ operativelylinked to a selected promoter sequence, rather than the protein itself,the amounts of DNA to be delivered and the routes of delivery mayparallel the protein amounts for vaccine or therapeutic deliverydescribed above and may also be determined readily by one of skill inthe art.

Thus, as one preferred example, a therapeutic composition may beformulated to contain a carrier or diluent and one or more plasmid orDNA molecule or recombinant virus containing a nucleic acid sequencewhich is anti-sense to the omp85 gene sequence (SEQ ID NO: 1 or 3), afragment thereof, under control of suitable sequences regulating theexpression thereof. In compositions containing the anti-sense omp85nucleic acid sequences, vehicles suitable for delivery of DNA may beemployed.

Additionally, these therapeutic compositions may be polyvalent. Suchcompositions may contain therapeutic components which are anti-sensesequences of other bacterial or viral origin, e.g., the anti-sensesequence of components of bacterial species homologous to Neiserriae orheterologous thereto and/or anti-sense sequence derived from antigensfrom a disease-causing virus. Depending upon the compatibility of thecomponents, the anti-sense sequences to the Omp85 antigens of thisinvention may thus be part of a multi-component therapeutic compositiondirected at more than a single disease.

As a further embodiment of this invention, a therapeutic method involvestreating a human or an animal for infection with N. gonorrhoeae or N.meningitidis by administering an effective amount of such a therapeuticnucleic-acid containing composition. An “effective amount” of a nucleicacid composition may be calculated as that amount capable of expressingin vivo the above effective amounts of exogenously delivered proteins.Such amounts may be determined by one of skill in the art. Preferably,such a composition is administered parenterally, preferablyintramuscularly or subcutaneously. However, it may also be formulated tobe administered by any other suitable route, including orally ortopically. The selection of the route of delivery and dosage of suchtherapeutic compositions is within the skill of the art.

As another example, a vaccine composition of this invention may be a DNAvaccine, which includes the omp85 DNA sequence (SEQ ID NOS: 1 or 3) or afragment thereof which encodes an immunogenic protein or peptide,optionally under the control of regulatory sequences. In one embodiment,this vaccine composition may contain a nucleic acid sequence thatencodes one or more of the isolated, recombinant, modified or multimericforms of the Omp85 antigen of the invention, or mixtures thereof.

In another embodiment of this invention, polyvalent vaccine compositionsmay include not only the nucleic acid sequence encoding the Omp85antigen or an immunogenic fragment thereof, but may also include nucleicacid sequences encoding antigens from other disease-causing agents. Suchother agents may be antigens from other Neisseriae strains. Such otheragents may be nucleic acid sequences encoding antigens from completelydistinct bacterial pathogens or from viral pathogens. Combinations ofthe antigen-encoding sequences of this invention with other nucleic acidsequences encoding antigens or fragments thereof from other pathogensare also encompassed by this invention for the purpose of inducing aprotective immune response in the vaccinated subject to more than asingle pathogen. The selection of these other vaccine components is nota limitation of the present invention, and may be left to one of skillin the art.

Such “DNA” or nucleic acid vaccines may include exemplary carriers asdescribed above for therapeutic compositions and, where suitable, thecomponents or additives described above with reference to theproteinaceous compositions, e.g. adjuvants, preservatives, chemicalstabilizers, etc. as well as other conventionally employed vaccineadditives. Additives suitable for use in nucleic acid compositions areknown to those of skill in the art, including certain lipids andliposomes, among other known components.

Generally, a suitable nucleic acid-based treatment contains between1×10⁻³ plaque forming unit (pfu) to 1×10¹² pfu per dose, if a virus isthe delivery vector. Otherwise, the dosage is adjusted to provide thesame amount of expressed protein as is provided by the protein vaccines.However, the dose, timing and mode of administration of thesecompositions may be determined by one of skill in the art. Such factorsas the age, and physical condition of the vaccinate may be taken intoaccount in determining the dose, timing and mode of administration ofthe immunogenic or vaccine composition of the invention.

VII. Drug Screening and Development

The proteins, antibodies and polynucleotide sequences of the presentinvention may also be used in the screening and development of chemicalcompounds or proteins which have utility as therapeutic drugs orvaccines for the treatment or diagnosis or prevention of diseases causedby infection with N. gonorrhoeae or N. meningitidis, and possibly forthe other microorganisms having homologous proteins to Omp85. As oneexample, a compound capable of binding to Omp85 and preventing itsbiological activity may be a useful drug component for the treatment orprevention of such non-symptomatic gonococcal infection or symptomaticdiseases as gonorrhea and non-symptomatic meningococcal infection andsymptomatic disease, e.g., spinal meningitis, among others. The methodsdescribed herein may also be applied to fragments of Omp85.

Suitable assay methods may be readily determined by one of skill in theart. Where desired, and depending on the assay selected, the selectedantigen(s), e.g., Omp85 or fragment thereof, may be immobilized directlyor indirectly (e.g., via an Omp85 antibody) on a suitable surface, e.g.,in an ELISA format. Such immobilization surfaces are well known. Forexample, a wettable inert bead may be used. Alternatively, the selectedantigen, e.g., Omp85, may be used in screening assays which do notrequire immobilization, e.g., in the screening of combinatoriallibraries. Assays and techniques exist for the screening and developmentof drugs capable of binding to an antigen of this invention, e.g.,Omp85. These include the use of phage display system for expressing theantigenic protein(s), and using a culture of transfected E. coli orother microorganism to produce the proteins for binding studies ofpotential binding compounds. See, for example, the techniques describedin G. Cesarini, FEBS Letters, 307(1):66-70 (July 1992); H. Gram et al.,J. Immunol. Meth., 161:169-176 (1993); C. Summer et al., Proc. Natl.Acad. Sci., USA, 89:3756-3760 (May 1992), incorporated by referenceherein.

Other conventional drug screening techniques may be employed using theproteins, antibodies or polynucleotide sequences of this invention. Asone example, a method for identifying compounds which specifically bindto a protein of this invention, e.g., Omp85, can include simply thesteps of contacting a selected Omp85 protein with a test compound topermit binding of the test compound to Omp85; and determining the amountof test compound, if any, which is bound to the Omp85 protein. Such amethod may involve the incubation of the test compound and the Omp85protein immobilized on a solid support. Similar methods may be employedfor one or more of the cassette string proteins.

Typically, the surface containing the immobilized ligand is permitted tocome into contact with a solution containing the protein and binding ismeasured using an appropriate detection system. Suitable detectionsystems include the streptavidin horse radish peroxidase conjugate,direct conjugation by a tag, e.g., fluorescein. Other systems are wellknown to those of skill in the art. This invention is not limited by thedetection system used.

Another method of identifying compounds which specifically bind to Omp85or another protein of this invention can include the steps of contactingthe protein, e.g., Omp85, immobilized on a solid support with both atest compound and the protein sequence which is a receptor for Omp85 topermit binding of the receptor to the Omp85 protein; and determining theamount of the receptor which is bound to the Omp85 protein. Theinhibition of binding of the normal protein by the test compound therebyindicates binding of the test compound to the Omp85 protein.

Still other conventional methods of drug screening can involve employinga suitable computer program to determine compounds having similar orcomplementary chemical structures to that of the Omp85 proteins (SEQ IDNOS: 2 and 4), and screening those compounds for competitive binding toOmp85. Such programs include the GRID program available from OxfordUniversity, Oxford, UK. (P. J. Goodford, “A Computational Procedure forDetermining Energetically Favorable Binding Sites on BiologicallyImportant Macromolecules”, J. Med. Chem., 28:849-857 (1985)); the MCSSprogram available from Molecular Simulations, Burlington, Mass. (A.Miranker and M. Karplus, “Functionality Maps of Binding Sites: AMultiple Copy Simultaneous Search Method”, Proteins: Structure, Functionand Genetics, 11:29-34 (1991)); the AUTODOCK program available fromScripps Research Institute, La Jolla, Calif. (D. S. Goodsell and A. J.Olsen, “Automated Docking of Substrates to Proteins by SimulatedAnnealing”, Proteins: Structure, Function, and Genetics, 8:195-202(1990)); and the DOCK program available from University of California,San Francisco, Calif. (I. D. Kuntz et al., “A Geometric Approach toMacromolecule-Ligand Interactions”, J. Mol. Biol., 161:269-288 (1982)).Additional commercially available computer databases for small molecularcompounds include Cambridge Structural Database, Fine Chemical Database,and CONCORD database. For a review see Rusinko, A., Chem. Des. Auto.News, 8:44-47 (1993).

Thus, the invention provides a method of identifying a pharmacomimeticof Omp85 of N. gonorrhoeae or N. meningitidis by using a combination ofsteps including identifying a compound which binds to Omp85 by screeningsaid Omp85 against a battery of compounds. Computer modelling of thethree dimensional structure of Omp85 or of the previously identifiedbinding compound permits the identification of a compound with the samethree dimensional structure as either Omp85 or its binding compound. Thecompound selected from these tests is then screened for the biologicalactivity of Omp85 or of a compound that binds Omp85, such as thedevelopment of antisera effective in the assay of Example 8 orcompetitive effect in that assay, respectively.

Thus, through use of such methods, the present invention is anticipatedto provide compounds capable of interacting with Omp85 or portionsthereof, and either enhancing or decreasing its biological activity, asdesired. Such compounds are believed to be encompassed by thisinvention.

The following examples are provided to illustrate the invention and donot limit the scope thereof. One skilled in the art will appreciate thatalthough specific reagents and conditions are outlined in the followingexamples, modifications can be made which are meant to be encompassed bythe spirit and scope of the invention.

Example 1 Bacterial Strains, Cells, Methods and Culture Conditions

N. gonorrhoeae strains FA19, FA635, FA1090, JS1, F62 and MS11LosA usedin the examples below were provided by Dr. William M. Shafer (EmoryUniversity, Atlanta, Ga.) Dr. John Swanson (Rocky Mountain Laboratories,Hamilton, Mont.) or the American Type Culture Collection 10801University Boulevard, Manassas, Va. 20110-2209. N. meningitidis strainsMP78, MP3, MP81, and HH were provided by Dr. Mark S. Peppler (Universityof Alberta, Edmonton, Alberta, Canada) and Dr. Zell McGee (University ofUtah, Salt Lake City, Utah). All other strains were acquired from theAmerican Type Culture Collection. Moraxella catarrhalis (ATCC# 8193) andNeisserial strains were grown on clear gonococcal typing media (SwansonJ., Infect. Immun., 19: 320-331 (1978)). E. coli and all other Gramnegative strains were grown an Luria broth.

E. coli XL1-Blue and SOLR cells were obtained from Stratagene CloningSystems (La Jolla, Calif.). DNA fragments were purified from agarosegels using GENE CLEAN™ II (Bio101, La Jolla, Calif.). Plasmids werepurified using the QIAPREP™ quick spin kit (Qiagen, Chatsworth, Calif.)Immunoblotting was done with MILLIPORE IMMOBILON™ PVDF reagent (Bedford,Mass.). Unless specified otherwise in the examples below, all reagentswere obtained from Sigma Chemical Co. (St. Louis, Mo.).

Example 2 Cloning and Immunological Screening of a Gonococcal GenomicLibrary

A genomic DNA library was produced by purifying N. gonorrhoeae strainFA19 genomic DNA and partially digesting the DNA into fragments with therestriction endonuclease Tsp509 (New England Biolabs). These DNAfragments were ligated with T4 DNA ligase (New England Biolabs) into theEcoRI restriction site of the lambda ZAP II™ bacteriophage vector(Stratagene Cloning Systems, La Jolla, Calif.). The ligated phage DNAwas packaged, and plated on E. coli DH5a (Gibco BRL, Gaithersburg, Md.)host cells. The resulting library was screened immunologically for theexpression of gonococcal surface proteins according to protocolsprovided with the Lambda ZAP II™ vector system. The plaques from thelibrary were screened with anti-GC-OM, an antiserum raised to isolatedgonococcal outer membranes.

Plasmid DNA was rescued from phage producing immunoreactive plaques. Theplasmid, pDR4, contained a 2.6 kbp gonococcal DNA insert. The gonococcalDNA in pDR4 was subcloned into pUP1 (Elkins C., J. Bacteriol., 173:3911-3913 (1991), generously provided by Dr. Chris Elkins (University ofNorth Carolina, Chapel Hill, N.C.) yielding pOmp85.

The cloned gonococcal DNA fragment in pOmp85 was characterized byrestriction enzyme analysis. The fragment contained three internalHincII restriction sites which allowed the subcloning of three fragmentsinto PBluescript™ plasmid (Stratagene Cloning Systems, La Jolla,Calif.). These fragments and pDR4 were sequenced by the University ofMontana Molecular Biology Facility. A gene-walking strategy was used tosequence those regions which could not be sequenced with universalvector primers. The entire length of the gonococcal DNA fragment wassequenced at least twice and most of the DNA was sequenced from bothstrands. The deduced amino acid sequence and comparisons of Omp85homologs were obtained with the MACVECTOR™ software package (EastmanChemical Co., New Haven, Conn.).

The 2.6 kb gonococcal DNA in pOmp85 was found to contain a large openreading frame (ORF) which encoded a polypeptide of 792 amino acids witha predicted molecular weight of 87.8 kDa (FIGS. 2A-2C) (SEQ ID NO:2).The polypeptide contained a putative signal peptide (Von Heijne G., Nuc.Acids Res., 14: 4683-4690 (1986)). Removal of this signal peptideyielded a mature polypeptide with an 85.8 kDa predicted molecularweight. The polypeptide also possesses a carboxyl-terminal phenylalanineresidue characteristic of outer membrane proteins (Struyve M. et al., J.Mol. Biol., 218: 141-148 (1991)). A ribosome-binding sequence precededthe initiation codon of the ORF. Potential promoter sequences were notreadily apparent. The ORF was preceded and followed by putativerho-independent transcriptional stop sites. The approximately 85 kDagonococcal protein expressed by pOmp85 was designated Omp85 and the geneencoding it was designated omp85.

Example 3 Cloning and Sequencing of Meningococcal OMP85

The meningococcal omp85 was obtained by PCR amplification using theBoehringer Mannheim EXPAND HIGH FIDELITY™ PCR System (Indianapolis,Ind.). The design of PCR primers was based on gonococcal omp85 andflanking sequences. The positive-sense omp85 PCR primer contained thefirst five codons of the gonococcal omp85 with an EcoRI restriction siteand two extra nucleotides added to the 5′ end (CGGAATTCATGAAACTGAAACAG)(SEQ ID NO: 5). The negative-sense omp85 PCR primer contained thereverse-compliment of six codons (TTGCAGTTTTTGCAATTC) (SEQ ID NO: 6) ofthe gonococcal ompH sequence located 244 base pairs 3′ of omp85termination codon. These primers were used in a PCR reaction withpurified N. meningitidis HH DNA as template. The meningococcal omp85 PCRproduct was ligated into pUP1 to yield pMCOmp85. The sequence of themeningococcal omp85 was obtained essentially as described for thegonococcal omp85.

The meningococcal omp85 was found to encode a 797 amino acid polypeptidewith a predicted molecular weight of 88.5 kDa (FIG. 5). Themeningococcal omp85 was 95% identical and 98% similar to gonococcalomp85. Between amino acid residues 720 and 745, the menigococcal Omp85varied substantially from gonococcal Omp 85, including the insertion offive additional amino acids.

Example 4 Presence of OMP85 in Strains of N. Gonorrhoeae and N.Meningitidis

A. Western Analysis

The Western blot analyses were performed as follows. Bacterial proteinswere separated by SDS-PAGE (12.5%) (Laemmli U K, Nature, 227: 680-695(1970)). The separated proteins were electrophoretically transferredonto PVDF as previously described (Judd R C., Anal. Biochem., 173:307-316 (1988)). Blotting was performed in 20 mM sodium phosphatebuffer, pH 8.0 for 2 hr at 600 mA. The PVDF membrane was blocked for 1hr with PBS Tween at room temperature, incubated overnight in anti-GC-OMor anti-Omp85 sera at 4° C., washed several times, incubated withprotein A-horseradish peroxidase conjugate (Boehringer Mannheim,Indianapolis, Ind.) and developed with 4-chloro-1-naphthol.

Western blot analysis of the proteins from E. coli DH5a/pOmp85 wasperformed by separating bacterial cell lysates by SDS-PAGE, stainingwith COOMASSIE BRILLIANT BLUE™ (CBB) stain or transferring the lysatesto membranes and probing with anti-GC-OM serum. The results revealedexpression of an approximately 85 kDa polypeptide that was reactive withthe anti-GC-OM serum (FIG. 1).

Anti-GC-OM serum was reacted in Western blot analysis with total cellproteins from six representative strains of N. gonorrhoeae and fourstrains of N. meningitidis. Whole cell lysates of E. coli DH5a, E. coliDH5a/pOmp85, and N. gonorrhoeae strains FA19, FA635, FA1090, JS1, F62and MS11LosA and N. meningitidis strains MP78, MP3, MP81 and HH wereseparated by 12.5% SDS-PAGE, blotted and probed with the anti-GC-OMserum. An immunoreactive, approximately 85 kDa protein band was detectedin all strains of N. gonorrhoeae and N. meningitidis (FIG. 3), but notin the control E. coli DH5a.

B. Southern Analysis

The Southern analyses are performed as follows. Chromosomal DNA wasobtained by phenol extraction (Moore D., “Preparation and analysis ofDNA”, in: Ausubel F M, et al., eds. Current Protocols in MolecularBiology. New York: John Wiley and Sons, (1997): 2.1.1-2.1.3) and thendigested with various endonucleases. Digested DNA waselectrophoretically separated on a 1% agarose gel and the DNAtransferred to nitrocellulose with the BIO-RAD™ Model 785 vacuum blotteraccording to instructions and Southern (Southern E., J. Mol. Biol., 98:503-510 (1975)). Probe DNA was extracted from agarose gels using Bio 101glassmilk (Vista, Calif.). The probe was labeled and the blot probedwith the AMERSHAM ECL™ system (Arlington Heights, Ill.).

A Southern blot (FIG. 4) illustrated the identification of omp85 in N.gonorrhoeae and N. meningitidis by Southern analysis. Genomic DNA fromE. coli DH5a, N. gonorrhoeae FA19 and N. meningitidis strains MP3, MP73,MP81 and HH were digested with restriction endonucleases (HincII, EcoRI,PstI, ClaI). Blots of the separated DNA digests were probed with a 688by fragment of gonococcal omp85 that extended from the most 3′ HincIIsite of omp85 to a HincII site in the vector near the 3′ end of thecloned gene. This fragment was used as a positive control.

In N. gonorrhoeae strain FA19 and in all of the N. meningitidis strains,the omp85 probe hybridized with a single DNA band. A single band wasalso identified in gonococcal strains FA635, FA1090, JS1, F62, and MS11(data not shown). These results suggested that omp85 was conserved as asingle copy in pathogenic Neisseria. Sequence data indicated that HincIIcleaved omp85 internally at three sites. The probe hybridized to afragment larger than itself in the HincII digest of gonococcal DNA(FA19-HincII). The genomic fragment resulted from an internal andexternal HincII cleavage; the probe lacked the flanking gonococcalsequence containing the external HincII site. The genomic HincII bandthat hybridized to the probe was probably derived from a single copy ofthe omp85 gene since it is unlikely that duplicate copies of the genewould have identically-located HincII sites, generating fragments ofidentical size.

The enzyme PstI cleaved 309 base pairs from the 5′ end of omp85. Thesingle fragment of PstI-digested gonococcal genomic DNA (FA19-PstI)which hybridized with the probe probably contained a single copy of thegene. It is possible, but unlikely, that the PstI fragments containedapproximately 2 kbp segments of omp85 in inverted orientations at eachend of the approximately 8 kbp fragment. Sequence data indicated thatthe enzymes ClaI and EcoRI did not cleave within omp85. The omp85 probehybridized to single bands of similar size in the ClaI digested DNA ofboth N. gonorrhoeae and N. meningitidis. These bands suggested a singlecopy of omp85, since it is unlikely that bands this small (<6 kbp)contained two copies of the approximately 2.3 kbp gene and it isunlikely that the bands represent fragments of identical size fromduplicate copies of the gene. Similar results were obtained forgonococcal strains FA635, FA1090, JS1, F62 and MS11 (data not shown).These results support the conclusion that omp85 is conserved as a singlecopy in pathogenic Neisseria.

Example 5 Similarity to Known Proteins

The non-redundant Genbank CDS database was searched (Altchul S D. etal., J. Mol. Biol., 215: 403-407 (1990)) for proteins similar togonococcal Omp85. The H. influenzae D-15-Ag was 31.5% identical and61.4% similar (identical plus conserved) to gonococcal Omp85. The P.multocida Oma87 was 31.6% identical and 61.3% similar to Omp85. Severalhypothetical proteins with similarity to Omp85 were identified. ABrucella abortus hypothetical protein (Genbank accession #U51683,Bearden, et al., unpublished) was 24.3% identical and 54.2% similar. Ahypothetical E. coli protein (Genbank accession #U70214, Schramm, etal., unpublished) was 33% identical and 62% similar to Omp85. Thegonococcal Omp85 was also similar to hypothetical proteins ofHelicobacter pylori (Genbank assession # AE001178—Tomb J F. et al.,Nature, 388: 539-547 (1997)), 23% identical and 51% similar, andBorrelia burgdorferi (Genbank assession #AE001178—Fraser C M. et al.,Nature, 390: 580-586 (1997)), 18% identical and 46% similar.

Example 6 Gene Organization and Flanking Genes

Several hundred base pairs of the DNA flanking the omp85 ORFs of both N.gonorrhoeae and N. meningitidis were sequenced. For both N. gonorrhoeaeand N. meningitidis, a gene similar to ompH of Salmonella typhimurium(Kosk P. et al., J. Biol. Chem., 264:18973-18980 (1989)) was identifiedapproximately 65 base pairs 3′ of the omp85 ORF. A gene similar to ompHhas also been identified in the same relative position in H. influenzae(Fleischmann, R D et al., Science, 269: 496-512 (1995)) and P.multocida. In H. influenzae, a gene encoding a hypothetical protein,HI0918, was located 5′ of the D-15-Ag gene (Fleischmann, R D et al,Science, 269: 496-512 (1995)). A gene homologous to the HI0918 gene wasidentified at the same relative position in N. gonorrhoeae. Theseresults indicated that the gene arrangement around omp85 homologs wasnotably conserved.

Example 7 Presence In Neisseriae and Other Species

Western blot analysis was used to determine if proteins similar to Omp85were produced by commensal Neisseriae and by other Gram negativespecies. To allow more specific immunological analysis, anOmp85-specific polyvalent rabbit sera was produced through use of afusion protein in which the first 200 amino acids of the gonococcalOmp85 were genetically fused to maltose binding protein (MBP).

A. Production of a MBP/Omp85 Fusion Protein

Fragments of gonococcal Omp85 were genetically fused to MBP, affinitypurified and used to produce Omp85-specific antiserum. Tsp5091 digestedomp85 from pOmp85 was ligated into the EcoRI digested, maltose bindingprotein fusion vector, pMAL-c2 (New England Biolabs, Beverly, Mass.).Sequence analysis had revealed that this could result in the fusion ofseveral different omp85 fragments in frame with malE in pMAL-c2. Theligated DNA was transformed into E. coli DH5a and the transformants werescreened for the expression of Omp85 antigens with the anti-OM serum. Anumber of immunoreactive clones were identified and characterized. Theplasmid in the most immunoreactive of these was designated pMO4 anddetermined by sequence analysis to express a fusion of MBP with thefirst 181 amino acids of Omp85. The MBP/Omp85 fusion protein wasaffinity purified from E. coli DH5a/pMO4 as previously described(Marchion D C et al, Mol. Microbiol., 6: 231-240 (1997)).

B. Raising of Anti-Sera

Purified MBP/Omp85 was used to raise an anti-Omp85 sera in New Zealandwhite rabbits. The rabbits were initially immunized with 1 mg of proteinin Freund's complete adjuvant administered subcutaneously. Two weekslater 1 mg was administered subcutaneously in Freund's incompleteadjuvant. The rabbits then received intravenous injections of 0.1 mgevery two weeks for eight weeks. Sera was collected and absorbed (1:1)with a lysed culture of E. coli/pMal-c2 induced with 1 mM IPTG for 1hour. The resulting rabbit serum was designated anti-Omp85.

C. Western Analysis of Representative Strains of N. gonorrhoeae and N.Meningitidis

The anti-Omp85 serum was used to probe Western blots containing celllysates of E. coli DH5a, E. coli DH5a/pOmp85 and representative strainsof N. gonorrhoeae and N. meningitidis (FIG. 6). Whole cell lysates of E.coli DH5a, E. coli DH5a/pOmp85, N. gonorrhoeae strains FA19, FA635,FA1090, JS1, F62 and MS11LosA, N. meningitidis strains MP78, MP3, MP81and HH, and E. coli DH5a/pMCOmp85 were separated by 12.5% SDS-PAGE,blotted and probed with the anti-Omp85 serum.

The anti-Omp85 serum reacted with the recombinant Omp85 produced by E.coli DH5a/pOmp85 and with proteins of approximately 85 kDa in all of theN. gonorrhoeae and N. meningitidis strains tested. These resultsindicated that Omp85 was conserved in the pathogenic Neisseriae.Presorption of the antiserum removed the majority of non-specificantibodies, but complete removal of all reactive antibody is notpossible, thus, some reactive bands can be seen in the E. coli lanes ofFIG. 6. Reactive bands in the 35 kDa-42 kDa range in N. gonorrhoeaelanes are Por proteins, which non-specifically bind protein-A.Meningococcal Pors do not bind protein-A as strongly (data not shown).

D. Western Analysis of Commensal Nesseriae and Other Gram NegativeSpecies

The anti-Omp85 was also used to probe Western blots containing celllysates of commensal Nesseriae and several other Gram negative species.Whole cell lysates of E. coli DH5a, E. coli DH5a/pOmp85, N. gonorrhoeaeFA19 (A), Neisseria pharyngis (A), Neisseria cinerea (A), Neisserialactamica (B), Neisseria mucosae (B), Neisseria flavescens (C),Neisseria animalis (C), Neisseria denitrificans (C), Moraxellacatarrhalis (D), Klebsiella pneumoniae, Pseudomonas aeruginosa, and N.meningitidis HH (A) were separated in a 12.5% SDSaPAGE gel, blotted andprobed with anti-Omp85. In a separate experiment, whole cell lysates ofE. coli DH5a, E. coli DH5a/pOmp85, N. gonorrhoeae FA19, Salmonellatyphimurium, Shigella flexneri, E. coli strains 35150(enterohemorrhagic—EHEC), 35401 (enterotoxigenic—ETEC), 43887(enteropathogenic—EPEC), 43892 (enteroinvasive—EIEC) and N. meningitidiswere separated in a 12.5% SDS-PAGE gel, blotted and probed withanti-Omp85.

Two SDS-PAGE (FIGS. 7A and 7B) illustrate the distribution of Omp85 inpathogenic and commensal Neisseria (relationship areas A, B, C, and D)and related Gram negative bacteria. Omp85 homologs were identified inall of the commensal Neisserial species tested. This suggested thatOmp85 was conserved among all the Neisserial species. The anti-Omp85serum failed to identify any Omp85 homologs in Moraxella catarrhaliswhich is closely related to the Neisseriae (Rossau R. et al, Int. J.Syst. Bacteriol., 39: 185-198 (1989)). Southern analysis confirmed theabsence of an omp85 homolog in this species (data not shown). The serumfailed to identify any Omp85 homologs in Klebsiella pneumoniae,Pseudomonas aeruginosa, Salmonella typhimurium, Shigella flexneri andfour pathogenic strains of E. coli tested.

Example 8 Gonococcal Cell Adherence Assay

Gonococcal cell adherence assays were performed to evaluate the effectof antiserum specific for outer membrane protein 85 (Omp85) on theability of gonococcal strains MS11LOSA (MS11) and FA19 to bind to Changepithelial cells. Fab fragments were prepared from antiserum to thefirst 178 amino acids of Omp85 (SEQ ID NO: 2), hyperimmune antiserum tobovine serum albumin (BSA) and normal rabbit serum (NRS) and added at 1μg, 10 μgs or 100 μgs per ml to wells containing a confluent layer ofChang conjunctiva cells. Approximately 2.5×10⁵ bacteria (N. gonorrhoeaestrain MS11 or FA19) were added to each well and allowed to adhere for 3hours. Following fixation and immunogold/silver staining, the number ofadherent gonococci was determined for 22 cells. The lowest and highestnumbers were discarded and the average number of bacteria/cell weredetermined. The resulting data is reported in the bar graph of FIG. 8,indicating that Omp85-specific antibody was able to bind to the surfaceof the bacteria and interfere with the ability of the bacteria to adhereto the Chang epithelial cells.

All publications cited in this specification are indicative of the levelof skill of those in the art to which this application pertains and areincorporated herein by reference herein. While the invention has beendescribed with reference to a particularly preferred embodiment, it willbe appreciated that modifications can be made without departing from thespirit of the invention. Such modifications are intended to fall withinthe scope of the appended claims.

1. A method of producing an immunogenic composition comprising isolatinga recombinant polypeptide comprising an epitope of at least 8consecutive amino acids within the amino acid sequence of SEQ ID NO: 4,and providing said polypeptide with a pharmaceutically acceptablecarrier, wherein said composition induces antibodies in a mammal thatbind to said epitope of SEQ ID NO: 4 and that interfere with adherenceof Neisseria gonorrhoeae as measured by the gonococcal cell adherenceassay.
 2. The method of claim 1, wherein said composition comprises asecond polypeptide or protein.
 3. The method according to claim 2,wherein said second polypeptide or protein is an antigen from apathogenic bacterial species that is heterologous or homologous toNeisseria gonorrhoeae or Neisseria meningitidis.
 4. The method accordingto claim 1, wherein said composition further comprises an adjuvant.